Identifying cancer-associated leukocyte profiles using high-resolution flow cytometry screening and machine learning.

Background: Machine learning (ML) is a valuable tool with the potential to aid clinical decision making. Adoption of ML to this end requires data that reliably correlates with the clinical outcome of interest; the advantage of ML is that it can model these correlations from complex multiparameter data sets that can be difficult to interpret conventionally. While currently available clinical data can be used in ML for this purpose, there exists the potential to discover new "biomarkers" that will enhance the effectiveness of ML in clinical decision making. Since the interaction of the immune system and cancer is a hallmark of tumor establishment and progression, one potential area for cancer biomarker discovery is through the investigation of cancer-related immune cell signatures. Hence, we hypothesize that blood immune cell signatures can act as a biomarker for cancer progression. Methods: To probe this, we have developed and tested a multiparameter cell-surface marker screening pipeline, using flow cytometry to obtain high-resolution systemic leukocyte population profiles that correlate with detection and characterization of several cancers in murine syngeneic tumor models. Results: We discovered a signature of several blood leukocyte subsets, the most notable of which were monocyte subsets, that could be used to train CATboost ML models to predict the presence and type of cancer present in the animals. Conclusions: Our findings highlight the potential utility of a screening approach to identify robust leukocyte biomarkers for cancer detection and characterization. This pipeline can easily be adapted to screen for cancer specific leukocyte markers from the blood of cancer patient.

T cell receptor sharing by cytotoxic T lymphocytes facilitates efficient virus control.

A remarkable feature of the adaptive immune system is the speed at which small numbers of antigen-specific lymphocytes can mediate a successful immune response. Rapid expansion of T and B lymphocyte clones that have receptors specific for a particular antigen is one of the primary means by which a swift response is generated. Although much of this clonal expansion is caused by the division of antigen-specific cells, here we demonstrate an additional mechanism by which the pool of effector T cells against a viral infection can quickly enlarge. Our data show that virus-specific CD8+ cytotoxic T lymphocytes (CTL) can transfer their T cell receptors (TCR) to recipient CTL of an unrelated specificity that, as a consequence, gain the antigen specificity of the donor T cell. This process occurs within minutes via membrane exchange and results in the recipient CTL acquiring the ability to recognize and eliminate cells targeted by the donor TCR, while still retaining the antigen specificity of its own TCR. Such receptor sharing allows rapid, proliferation-independent expansion of virus-specific T cell clones of low frequency and plays a highly significant antiviral role that can protect the host from an otherwise lethal infection.

Identification of a novel antigen cross-presenting cell type in spleen.

Antigen-presenting cells (APC), like dendritic cells (DC), are essential for T-cell activation, leading to immunity or tolerance. Multiple DC subsets each play a unique role in the immune response. Here, a novel splenic dendritic-like APC has been characterized in mice that has immune function and cell surface phenotype distinct from other, described DC subsets. These were identified as a cell type continuously produced in spleen long-term cultures (LTC) and have an in vivo equivalent cell type in mice, namely 'L-DC'. This study characterizes LTC-DC in terms of marker phenotype and function, and compares them with L-DC and other known splenic DC and myeloid subsets. L-DC display a myeloid dendritic-like phenotype equivalent to LTC-DC as CD11c(lo) CD11b(hi) MHC-II(-) CD8α(-) cells, distinct by high accessibility and endocytic capacity for blood-borne antigen. Both LTC-DC and L-DC have strong antigen cross-presentation ability leading to strong activation of CD8(+) T cells, particularly after exposure to lipopolysaccharide. However, they have weak ability to stimulate CD4(+) T cells in antigen-specific responses. Evidence is presented here for a novel DC type produced by in vitro haematopoiesis which has distinct antigen-presenting potential and reflects a DC subset present also in vivo in spleen.

Bystander B cells rapidly acquire antigen receptors from activated B cells by membrane transfer.

The B cell antigen receptor (BCR) efficiently facilitates the capture and processing of a specific antigen for presentation on MHC class II molecules to antigen-specific CD4(+) T cells (1). Despite this, the majority of B cells are thought to play only a limited role in CD4(+) T cell activation because BCRs are clonotypically expressed. Here, we show, however, that activated B cells can, both in vitro and in vivo, rapidly donate their BCR to bystander B cells, a process that is mediated by direct membrane transfer between adjacent B cells and is amplified by the interaction of the BCR with a specific antigen. This results in a dramatic expansion in the number of antigen-binding B cells in vivo, with the transferred BCR endowing recipient B cells with the ability to present a specific antigen to antigen-specific CD4(+) T cells.

Custom-designed Small Animal focal iRradiation Jig (SARJ): design, manufacture and dosimetric evaluation.

OBJECTIVE: Preclinical animal models allow testing and refinement of novel therapeutic strategies. The most common preclinical animal irradiators are fixed source cabinet irradiators, which are vastly inferior to clinical linear accelerators capable of delivering highly conformal and precise treatments. The purpose of this study was to design, manufacture and test an irradiation jig (small animal focal irradiation jig, SARJ) that would enable focal irradiation of subcutaneous tumours in a standard fixed source cabinet irradiator. METHODS AND MATERIALS: A lead shielded SARJ was designed to rotate animal holders about the longitudinal axis and slide vertically from the base plate. Radiation dosimetry was undertaken using the built-in ion chamber and GAFChromic RTQA2 and EBT-XD films. Treatment effectiveness was determined by irradiating mice with subcutaneous melanoma lesions using a dose of 36 Gy in three fractions (12 Gy x 3) over three consecutive days. RESULTS: The SARJ was tested for X-ray shielding effectiveness, verification of dose rate, total dose delivered to tumour and dose uniformity. Accurate and uniform delivery of X-ray dose was achieved. X-ray doses were limited to the tumour site when animal holders were rotated around their longitudinal axis to 15o and 195o, allowing sequential dose delivery using parallel-opposed tangential beams. Irradiation of subcutaneous melanoma tumour established on the flanks of mice showed regression. CONCLUSION: SARJ enabled delivery of tangential parallel-opposed radiation beams to subcutaneous tumours in up to five mice simultaneously. SARJ allowed high throughput testing of clinically relevant dose delivery using a standard cabinet-style fixed source irradiator. ADVANCES IN KNOWLEDGE: A custom designed jig has been manufactured to fit into conventional cabinet irradiators and is dosimetrically validated to deliver clinically relevant dose distributions to subcutaneous tumours in mice for preclinical studies.

Mycoplasma contaminants present in exosome preparations induce polyclonal B cell responses.

Exosome fractions of dendritic cells (DC) produced in long-term cultures (LTC) were found to contain Mycoplasma contaminants. In this study, Mycoplasma-infected, -uninfected, and -reinfected cultures of DC and control cell lines have been compared for their capacity to activate lymphocytes. Using differential centrifugation, size fractionation, and inhibition assays, it has been possible to map Mycoplasma to the exosome or vesicle fraction purified from culture supernatant (CSN). Mycoplasma fractions were shown to induce proliferation of B and not T cells. The B cell response was sensitive to mitomycin C and primaquine, both known antibiotics, but resistant to protease and DNase, suggesting a role for lipoproteins. Mycoplasma-contaminated exosome fractions of LTC-DC were potent mitogens for naive B cells and promoted Ig secretion. In contrast to the polyclonal B cell mitogen LPS, they were unable to promote Ig isotype switching. They induced polyclonal activation of all B cell subsets, including naive B cells, the T1 and T2 subsets of transitional B cells, marginal zone (MZ), and follicular (FO) B cells. The B cell proliferative response was not antigen-specific and occurred independently of T cell help. Implications for autoimmune sequelae associated with Mycoplasma infection are discussed along with the possibility that primaquine could be an effective treatment for Mycoplasma infection in humans. This study highlights the close association between exosomes and infectious agents like Mycoplasma and cautions about purification procedures for preparation of exosomes for studies on immunity.

Cytolytic DNA vaccine encoding lytic perforin augments the maturation of- and antigen presentation by- dendritic cells in a time-dependent manner.

The use of cost-effective vaccines capable of inducing robust CD8+ T cell immunity will contribute significantly towards the elimination of persistent viral infections and cancers worldwide. We have previously reported that a cytolytic DNA vaccine encoding an immunogen and a truncated mouse perforin (PRF) protein significantly augments anti-viral T cell (including CD8+ T cell) immunity. Thus, the current study investigated whether this vaccine enhances activation of dendritic cells (DCs) resulting in greater priming of CD8+ T cell immunity. In vitro data showed that transfection of HEK293T cells with the cytolytic DNA resulted in the release of lactate dehydrogenase, indicative of necrotic/lytic cell death. In vitro exposure of this lytic cell debris to purified DCs from naïve C57BL/6 mice resulted in maturation of DCs as determined by up-regulation of CD80/CD86. Using activation/proliferation of adoptively transferred OT-I CD8+ T cells to measure antigen presentation by DCs in vivo, it was determined that cytolytic DNA immunisation resulted in a time-dependent increase in the proliferation of OT-I CD8+ T cells compared to canonical DNA immunisation. Overall, the data suggest that the cytolytic DNA vaccine increases the activity of DCs which has important implications for the design of DNA vaccines to improve their translational prospects.

Alternatively activated macrophage possess antitumor cytotoxicity that is induced by IL-4 and mediated by arginase-1.

Earlier studies have shown that the adoptive transfer of Th2-polarized CD4 T cells can clear established tumors from mice in an antigen-specific manner. Although eosinophils were implicated in this process, the exact mechanism of tumor clearance and which immune effector cells were involved, remain to be defined. Consequently, experiments were undertaken to elucidate the mechanism of Th2-mediated destruction of B16-F1 melanoma cells by examining the in vitro antitumor activity of leukocytes within a type-2 inflammatory infiltrate. The experimental data show that activation of alternatively activated macrophages (aaMacs) within type-2 infiltrates by IL-4 or IL-13 can inhibit B16-F1 melanoma cell proliferation through a mechanism that is dependent on arginase-1 depletion of L-arginine within the tumor cell microenvironment. Interestingly, whilst at higher E:T ratios aaMac exhibited antitumor activity, at lower E:T ratios aaMacs were observed to enhance rather than inhibit B16-F1 melanoma cell growth. This highlights the fine balance between stimulating the antitumorigenic and protumorigenic properties of aaMacs in tumor immunotherapy protocols.

Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation.

The stable incorporation of the intracellular fluorescent dye 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) into cells provides a powerful tool to monitor cell migration, and to quantify cell division, because of the sequential decrease in fluorescent labeling in daughter cells. CFSE-labeled lymphocytes have been used to analyze the relationship between cell division and differentiation of cell function, and cell proliferation versus apoptosis, both in vivo and in vitro, and have allowed analysis of the site of response to antigens in vivo.

Exosomes secreted by bacterially infected macrophages are proinflammatory.

Exosomes are small vesicles that are secreted from cells. They are derived from multivesicular endosomes that fuse with the plasma membrane, thereby releasing their internal vesicles into the extracellular environment. Exosomes from antigen-presenting cells contain a range of immunostimulatory molecules that activate T cells, which suggests that they may have an important role in the propagation of immune responses. Of considerable interest is the finding that exosomes derived from bacterially infected macrophages carry bacterial coat components and use these to stimulate bystander macrophages and neutrophils to secrete proinflammatory mediators, including tumor necrosis factor-alpha, the chemokine regulated upon activation, normal T cell-expressed and -secreted (RANTES, also known as CCL5), and inducible nitric oxide synthase. Here, we address these studies in relation to other findings on dendritic cell-derived exosomes that are also powerful immunoregulators.

Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester.

This protocol outlines the carboxyfluorescein diacetate succinimidyl ester (CFSE) method for following the proliferation of human lymphocytes in vitro and mouse lymphocytes both in vitro and in vivo. The method relies on the ability of CFSE to covalently label long-lived intracellular molecules with the highly fluorescent dye, carboxyfluorescein. Following each cell division, the equal distribution of these fluorescent molecules to progeny cells results in a halving of the fluorescence of daughter cells. The CFSE labeling protocol described, which typically takes <1 h to perform, allows the detection of up to eight cell divisions before CFSE fluorescence is decreased to the background fluorescence of unlabeled cells. Protocols are outlined for labeling large and small numbers of human and mouse lymphocytes, labeling conditions being identified that minimize CFSE toxicity but maximize the number of cell divisions detected. An important feature of the technique is that division-dependent changes in the expression of cell-surface markers and intracellular proteins are easily quantified by flow cytometry.

The immunogenicity of dendritic cell-derived exosomes.

Exosome production represents an alternate endocytic pathway for secretion. Multivesicular endosomes (MVE) fuse with the plasma membrane expelling internal vesicles or exosomes from cells. Exosome production has been recently described for immune cells including B cells, dendritic cells (DC), mast cells, macrophages and T cells. Exosomes derived from some DC populations stimulate T lymphocyte proliferation in vitro and have potent capacity to generate anti-tumour immune responses in vivo. These reported studies have involved in vitro grown mature DC expanded from precursors with cytokines. However, immature DC produce higher numbers of exosomes than mature DC and this is thought to be due to a reduction in endocytosis as DC mature, associated with reduced reformation of MVE and reduced exosome formation. This lab pioneered a method to generate immature DC in spleen long-term cultures (LTC). DC produced in cultures represent immature myeloid DC, highly endocytic but with weak capacity to stimulate T cells. LTC-DC produce exosomes and contain many MVE. This prompted a study of immunogenic potential with a view to the potential use of exosomes in vaccination and immunotherapy. DC produced in cultures represent immature myeloid DC, highly endocytic but with weak capacity to stimulate T cells. Exosomes were isolated by differential centrifugation from LTC-DC and shown by marker expression to arise by budding from the LAMP-1+ limiting endosomal membrane of MVE. These LTC-derived exosomes appear however to lack immunostimulatory markers like CD86, CD40, MHC-I and MHC-II. While LTC-DC can stimulate antigen-specific proliferation of CD4+ T cells, exosome preparations derived from antigen-pulsed DC were unable to stimulate purified naïve T cells in vitro. They were however found to weakly activate allogeneic CD8+ T cells in vitro. Tumour antigen-pulsed LTC-DC or their exosomes could induce a protective response in mice against growth of a transplanted tumour but could not induce a response to clear an existing tumour. Exosomes derived from immature DC can modulate immune responses, but do not function in direct T cell activation in vitro. Modulation of immune responses by exosomes produced by immature DC may be dependent on the presence of other antigen presenting DC subsets in the animal. The possible function of immature DC and their exosomes in maintenance of tolerance and in the induction of immunity is discussed.

Maturation of function in dendritic cells for tolerance and immunity.

The capacity of antigen presenting dendritic cells (DC) to function in both tolerance and immunity is now well documented. The function and characteristics of different DC subsets are reviewed here and their capacity to activate T cells under different conditions of maturation and activation is discussed. The immunogenic potential of exosomes produced by DC is also considered in light of evidence that the capacity of exosomes to activate T cells for tolerance or immunity appears to mirror that of the parent DC. A model is proposed whereby exosomes produced by immature DC can function to maintain peripheral tolerance, while exosomes produced by more mature DC can stimulate effector T cells.