Mass Cytometry Defines Virus-Specific CD4 + T Cells in Influenza Vaccination

The antiviral response to influenza virus is complex and multifaceted, involving many immune cell subsets. There is an urgent need to understand the role of CD4+ T cells, which orchestrate an effective antiviral response, to improve vaccine design strategies. In this study, we analyzed PBMCs from human participants immunized with influenza vaccine, using high-dimensional single-cell proteomic immune profiling by mass cytometry. Data were analyzed using a novel clustering algorithm, denoised ragged pruning, to define possible influenza virus-specific clusters of CD4+ T cells. Denoised ragged pruning identified six clusters of cells. Among these, one cluster (Cluster 3) was found to increase in abundance following stimulation with influenza virus peptide ex vivo. A separate cluster (Cluster 4) was found to expand in abundance between days 0 and 7 postvaccination, indicating that it is vaccine responsive. We examined the expression profiles of all six clusters to characterize their lineage, functionality, and possible role in the response to influenza vaccine. Clusters 3 and 4 consisted of effector memory cells, with high CD154 expression. Cluster 3 expressed cytokines like IL-2, IFN-γ, and TNF-α, whereas Cluster 4 expressed IL-17. Interestingly, some participants had low abundance of Clusters 3 and 4, whereas others had higher abundance of one of these clusters compared with the other. Taken together, we present an approach for identifying novel influenza virus-reactive CD4+ T cell subsets, a method that could help advance understanding of the immune response to influenza, predict responsiveness to vaccines, and aid in better vaccine design

Analogue peptides for the immunotherapy of human acute myeloid leukemia

Accepted manuscript. The final publication is available at: http://link.springer.com/article/10.1007%2Fs00262-015-1762-9The use of peptide vaccines, enhanced by adjuvants, has shown some efficacy in clinical trials. However, responses are often short-lived and rarely induce notable memory responses. The reason is that self-antigens have already been presented to the immune system as the tumor develops, leading to tolerance or some degree of host tumor cell destruction. To try to break tolerance against self-antigens, one of the methods employed has been to modify peptides at the anchor residues to enhance their ability to bind major histocompatibility complex molecules, extending their exposure to the T-cell receptor. These modified or analogue peptides have been investigated as stimulators of the immune system in patients with different cancers with variable but sometimes notable success. In this review we describe the background and recent developments in the use of analogue peptides for the immunotherapy of acute myeloid leukemia describing knowledge useful for the application of analogue peptide treatments for other malignancies

Lower levels of Th1 and Th2 cytokines in cerebrospinal fluid (CSF) at the time of initial CSF shunt placement in children are associated with subsequent shunt revision surgeries

OBJECTIVE: We compare cytokine profiles at the time of initial CSF shunt placement between children who required no subsequent shunt revision surgeries and children requiring repeated CSF shunt revision surgeries for CSF shunt failure. We also describe the cytokine profiles across surgical episodes for children who undergo multiple subsequent revision surgeries. METHODS: This pilot study was nested within an ongoing prospective multicenter study collecting CSF samples and clinical data at the time of CSF shunt surgeries since August 2014. We selected cases where CSF was available for children who underwent an initial CSF shunt placement and had no subsequent shunt revision surgeries during \u3e=24 months of follow-up (n = 7); as well as children who underwent an initial CSF shunt placement and then required repeated CSF shunt revision surgeries (n = 3). Levels of 92 human cytokines were measured using the Olink immunoassay and 41 human cytokines were measured using Luminex based bead array on CSF obtained at the time of each child\u27s initial CSF shunt placement and were displayed in heat maps. RESULTS: Qualitatively similar profiles for the majority of cytokines were observed among the patients in each group in both Olink and Luminex assays. Lower levels of MCP-3, CASP-8, CD5, CXCL9, CXCL11, eotaxin, IFN-γ, IL-13, IP-10, and OSM at the time of initial surgery were noted in the children who went on to require multiple surgeries. Pro- and anti-inflammatory cytokines were selected a priori and shown across subsequent revision surgeries for the 3 patients. Cytokine patterns differed between patients, but within a given patient pro-inflammatory and anti-inflammatory cytokines acted in a parallel fashion, with the exception of IL-4. CONCLUSIONS: Heat maps of cytokine levels at the time of initial CSF shunt placement for each child undergoing only a single initial CSF shunt placement and for each child undergoing repeat CSF shunt revision surgeries demonstrated qualitatively similar profiles for the majority of cytokines. Lower levels of MCP-3, CASP-8, CD5, CXCL9, CXCL11, eotaxin, IFN-γ, IL-13, IP-10, and OSM at the time of initial surgery were noted in the children who went on to require multiple surgeries. Better stratification by patient age, etiology, and mechanism of failure is needed to develop a deeper understanding of the mechanism of inflammation in the development of hydrocephalus and response to shunting in children

SITC cancer immunotherapy resource document: a compass in the land of biomarker discovery.

Since the publication of the Society for Immunotherapy of Cancer\u27s (SITC) original cancer immunotherapy biomarkers resource document, there have been remarkable breakthroughs in cancer immunotherapy, in particular the development and approval of immune checkpoint inhibitors, engineered cellular therapies, and tumor vaccines to unleash antitumor immune activity. The most notable feature of these breakthroughs is the achievement of durable clinical responses in some patients, enabling long-term survival. These durable responses have been noted in tumor types that were not previously considered immunotherapy-sensitive, suggesting that all patients with cancer may have the potential to benefit from immunotherapy. However, a persistent challenge in the field is the fact that only a minority of patients respond to immunotherapy, especially those therapies that rely on endogenous immune activation such as checkpoint inhibitors and vaccination due to the complex and heterogeneous immune escape mechanisms which can develop in each patient. Therefore, the development of robust biomarkers for each immunotherapy strategy, enabling rational patient selection and the design of precise combination therapies, is key for the continued success and improvement of immunotherapy. In this document, we summarize and update established biomarkers, guidelines, and regulatory considerations for clinical immune biomarker development, discuss well-known and novel technologies for biomarker discovery and validation, and provide tools and resources that can be used by the biomarker research community to facilitate the continued development of immuno-oncology and aid in the goal of durable responses in all patients

Minimal information about T cell assays: the process of reaching the community of T cell immunologists in cancer and beyond

Many assays to evaluate the nature, breadth, and quality of antigen-specific T cell responses are currently applied in human medicine. In most cases, assay-related protocols are developed on an individual laboratory basis, resulting in a large number of different protocols being applied worldwide. Together with the inherent complexity of cellular assays, this leads to unnecessary limitations in the ability to compare results generated across institutions. Over the past few years a number of critical assay parameters have been identified which influence test performance irrespective of protocol, material, and reagents used. Describing these critical factors as an integral part of any published report will both facilitate the comparison of data generated across institutions and lead to improvements in the assays themselves. To this end, the Minimal Information About T Cell Assays (MIATA) project was initiated. The objective of MIATA is to achieve a broad consensus on which T cell assay parameters should be reported in scientific publications and to propose a mechanism for reporting these in a systematic manner. To add maximum value for the scientific community, a step-wise, open, and field-spanning approach has been taken to achieve technical precision, user-friendliness, adequate incorporation of concerns, and high acceptance among peers. Here, we describe the past, present, and future perspectives of the MIATA project. We suggest that the approach taken can be generically applied to projects in which a broad consensus has to be reached among scientists working in fragmented fields, such as immunology. An additional objective of this undertaking is to engage the broader scientific community to comment on MIATA and to become an active participant in the project

Ontogeny of Toll-Like Receptor Mediated Cytokine Responses of Human Blood Mononuclear Cells

Newborns and young infants suffer increased infectious morbidity and mortality as compared to older children and adults. Morbidity and mortality due to infection are highest during the first weeks of life, decreasing over several years. Furthermore, most vaccines are not administered around birth, but over the first few years of life. A more complete understanding of the ontogeny of the immune system over the first years of life is thus urgently needed. Here, we applied the most comprehensive analysis focused on the innate immune response following TLR stimulation over the first 2 years of life in the largest such longitudinal cohort studied to-date (35 subjects). We found that innate TLR responses (i) known to support Th17 adaptive immune responses (IL-23, IL-6) peaked around birth and declined over the following 2 years only to increase again by adulthood; (ii) potentially supporting antiviral defense (IFN-α) reached adult level function by 1 year of age; (iii) known to support Th1 type immunity (IL-12p70, IFN-γ) slowly rose from a low at birth but remained far below adult responses even at 2 years of age; (iv) inducing IL-10 production steadily declined from a high around birth to adult levels by 1 or 2 years of age, and; (v) leading to production of TNF-α or IL-1β varied by stimuli. Our data contradict the notion of a linear progression from an ‘immature’ neonatal to a ‘mature’ adult pattern, but instead indicate the existence of qualitative and quantitative age-specific changes in innate immune reactivity in response to TLR stimulation

Distinct predictive biomarker candidates for response to anti-CTLA-4 and anti-PD-1 immunotherapy in melanoma patients

Background: While immune checkpoint blockade has greatly improved clinical outcomes in diseases such as melanoma, there remains a need for predictive biomarkers to determine who will likely benefit most from which therapy. To date, most biomarkers of response have been identified in the tumors themselves. Biomarkers that could be assessed from peripheral blood would be even more desirable, because of ease of access and reproducibility of sampling. Methods: We used mass cytometry (CyTOF) to comprehensively profile peripheral blood of melanoma patients, in order to find predictive biomarkers of response to anti-CTLA-4 or anti-PD-1 therapy. Using a panel of ~ 40 surface and intracellular markers, we performed in-depth phenotypic and functional immune profiling to identify potential predictive biomarker candidates. Results: Immune profiling of baseline peripheral blood samples using CyTOF revealed that anti-CTLA-4 and anti-PD-1 therapies have distinct sets of candidate biomarkers. The distribution of CD4+ and CD8+ memory/non-memory cells and other memory subsets was different between responders and non-responders to anti-CTLA-4 therapy. In anti-PD-1 (but not anti-CTLA-4) treated patients, we discovered differences in CD69 and MIP-1β expressing NK cells between responders and non-responders. Finally, multivariate analysis was used to develop a model for the prediction of response. Conclusions: Our results indicate that anti-CTLA-4 and anti-PD-1 have distinct predictive biomarker candidates. CD4+ and CD8+ memory T cell subsets play an important role in response to anti-CTLA-4, and are potential biomarker candidates. For anti-PD-1 therapy, NK cell subsets (but not memory T cell subsets) correlated with clinical response to therapy. These functionally active NK cell subsets likely play a critical role in the anti-tumor response triggered by anti-PD-1. Electronic supplementary material The online version of this article (10.1186/s40425-018-0328-8) contains supplementary material, which is available to authorized users

Motility-related protein-1 (MRP-1/CD9) expression can predict disease-free survival in patients with squamous cell carcinoma of the head and neck

CD9 is a transmembrane protein that has been implicated in cell adhesion, motility and proliferation, and numerous studies have demonstrated the prognostic value of its expression in different solid tumours. The purpose of this study is to determine the predictive value of CD9 in squamous cell carcinoma (SCC) of the head and neck. A total of 153 cases were examined for CD9 expression using immunohistochemistry applied on formalin-fixed, paraffin-embedded tissue. Cases were stratified in two categories depending on CD9 expression, as positive (>/=50% positive cells) or reduced (<50%). In all, 108 cases were positive for CD9 (85 cases with membranous, and 23 with both membranous and cytoplasmic staining) and 45 reduced expression. Reduced CD9 expression was significantly associated with high grade (P=0.0007) and lower disease-free survival (DFS) (P=0.017). The latter retained its significance in the multivariate analysis. When the 23 cases with both membranous and cytoplasmic patterns were studied as a separate subgroup, there were significant associations between CD9 expression and tumour grade (P=0.025) (95% CI 11-68), tumour stage (P=0.08) (95% CI 3.5-86) and the occurrence of any failure (P=0.083) (95% CI -1.7-57). Immunohistochemical CD9 expression proved to be an independent prognostic factor in SCC of the head and neck, and it may detect patients at a high risk of recurrence. In addition, the cytoplasmic pattern seems to have an even more significant value. However, this finding is limited to the small number of cases with this pattern

Lymphocyte subsets in human immunodeficiency virus-unexposed Brazilian individuals from birth to adulthood

Ethnic origin, genetics, gender and environmental factors have been shown to influence some immunologic indices, so that development of reference values for populations of different backgrounds may be necessary. We have determined the distribution of lymphocyte subsets in healthy Brazilian individuals from birth to adulthood. Lymphocyte subsets were determined using four-colour cytometry in a cross-sectional study of 463 human immunodeficiency virus-unexposed children and adults from birth through 49 years of age. Lymphocyte subsets varied according to age, as previously observed in other studies. However, total CD4+ T cell numbers were lower than what was described in the Pediatric AIDS Clinical Trials Group P1009 (PACTG P1009), which assessed an American population of predominantly African and Hispanic backgrounds until the 12-18 year age range, when values were comparable. Naïve percentages and absolute values of CD8+ T cells, as assessed by CD45RA expression, were also lower than the PACTG P1009 data for all analysed age ranges. CD38 expression on both CD4+ and CD8+ T cells was lower than the PACTG P1009 values, with a widening gap between the two studies at older age ranges. Different patterns of cell differentiation seem to occur in different settings and may have characteristic expression within each population.Universidade Federal de São Paulo (UNIFESP) Departamento de MedicinaCentro Assistencial Cruz de MaltaUniversidade Federal de São Paulo (UNIFESP) Departamento de PediatriaUNIFESP, Depto. de MedicinaUNIFESP, Depto. de PediatriaSciEL

A systematic approach to biomarker discovery; Preamble to "the iSBTc-FDA taskforce on immunotherapy biomarkers"

The International Society for the Biological Therapy of Cancer (iSBTc) has initiated in collaboration with the United States Food and Drug Administration (FDA) a programmatic look at innovative avenues for the identification of relevant parameters to assist clinical and basic scientists who study the natural course of host/tumor interactions or their response to immune manipulation. The task force has two primary goals: 1) identify best practices of standardized and validated immune monitoring procedures and assays to promote inter-trial comparisons and 2) develop strategies for the identification of novel biomarkers that may enhance our understating of principles governing human cancer immune biology and, consequently, implement their clinical application. Two working groups were created that will report the developed best practices at an NCI/FDA/iSBTc sponsored workshop tied to the annual meeting of the iSBTc to be held in Washington DC in the Fall of 2009. This foreword provides an overview of the task force and invites feedback from readers that might be incorporated in the discussions and in the final document