Differential Uptake of Murine and Human Exosomes by Normal and Inflamed Peripheral Tissues and Brain

Background: Exosomes function as an intercellular communication system conveying messages from donor to target cells in nearby or distant tissues. Many aspects of exosome trafficking remain unresolved, however. Here, we investigated uptake of ten radiolabeled murine or human exosomes of various cellular origins by the liver, kidney, spleen, and lung of male CD-1 mice. Methods: We radioactively labeled 10 exosomes from mouse or human cancerous or non-cancerous lines, injected them intravenously into male CD-1 mice, and studied their tissue uptake. We examined the ability of wheatgerm agglutinin (WGA), mannose-6 phosphate (M6P), and inflammation induced by lipopolysaccharide (LPS) to modulate uptake. We measured uptake rate using multiple-time regression analysis and used heat mapping and path analysis to correlate tissue and exosomal influences on uptake. Results: Except for the uptake of SCCVII exosomes by kidney, all exosomes were taken up by all tissues, although the uptake levels varied broadly among exosomes and tissues. The liver/serum uptake ratio for exosomes from primary human T-cells was the highest at 4,500 mL/g. Species of origin (mouse vs human) or source (cancerous vs noncancerous cells) did not influence tissue uptake. The uptake of some exosomes was altered by WGA and LPS but not by M6P, except for uptake inhibition of J774A.1 exosomes by liver, suggesting use of the M6P receptor. WGA or LPS treatments enhanced uptake of exosomes by brain and lung but inhibited uptake by liver and spleen. Response to LPS was not, however, predictive of response to WGA. No evidence for a universal binding site controlling exosome uptake was obtained. Applying path analysis and heat map analysis to the data, including our published results for brain, we found that exosome uptake patterns for lung and brain responded similarly to WGA or to LPS, whereas those for liver and spleen clustered together. In path analysis, the 10 exosomes clustered into distinct groups, suggesting that their bindings sites are similarly clustered. Conclusions: Uptake of exosomes by peripheral tissues is differentially regulated by both exosomes and target tissues and is dependent on the number and types of mutually interactive binding sites.

Siglec-5 is an inhibitory immune checkpoint molecule for human T cells

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are a family of immunoglobulin-type lectins that mediate protein-carbohydrate interactions via sialic acids attached to glycoproteins or glycolipids. Most of the CD33-related Siglecs (CD33rSiglecs), a major subfamily of rapidly evolving Siglecs, contain a cytoplasmic signaling domain consisting of the immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) and mediate suppressive signals for lymphoid and myeloid cells. While most CD33rSiglecs are expressed by innate immune cells, such as monocytes and neutrophils, to date, the expression of Siglecs in human T cells has not been well appreciated. In this study, we found that Siglec-5, a member of the CD33rSiglecs, is expressed by most activated T cells upon antigen receptor stimulation. Functionally, Siglec-5 suppresses T cell activation. In support of these findings, we found that Siglec-5 overexpression abrogates antigen receptor induced activation of Nuclear factor of activated T cells (NFAT) and Activator protein 1 (AP-1). Furthermore, we show that GBS β-protein, a known bacterial ligand of Siglec-5, reduces the production of cytokines and cytolytic molecules by activated primary T cells in a Siglec-5 dependent manner. Our data also show that some cancer cell lines express a putative Siglec-5 ligand(s), and that the presence of soluble Siglec-5 enhances tumor-cell specific T cell activation, suggesting that some tumor cells inhibit T cell activation via Siglec-5. Together, our data demonstrate that Siglec-5 is a previously unrecognized inhibitory T cell immune checkpoint molecule and suggests that blockade of Siglec-5 could serve as a new strategy to enhance anti-tumor T cell functions.

Structural basis of HLX10 PD-1 receptor recognition, a promising anti-PD-1 antibody clinical candidate for cancer immunotherapy

Cancer immunotherapies, such as checkpoint blockade of programmed cell death protein-1 (PD-1), represents a breakthrough in cancer treatment, resulting in unprecedented results in terms of overall and progression-free survival. Discovery and development of novel anti PD-1 inhibitors remains a field of intense investigation, where novel monoclonal antibodies (mAbs) and novel antibody formats (e.g., novel isotype, bispecific mAb and low-molecular-weight compounds) are major source of future therapeutic candidates. HLX10, a fully humanized IgG4 monoclonal antibody against PD-1 receptor, increased functional activities of human T-cells and showed in vitro, and anti-tumor activity in several tumor models. The combined inhibition of PD-1/PDL-1 and angiogenesis pathways using anti-VEGF antibody may enhance a sustained suppression of cancer-related angiogenesis and tumor elimination. To elucidate HLX10’s mode of action, we solved the structure of HLX10 in complex with PD-1 receptor. Detailed epitope analysis showed that HLX10 has a unique mode of recognition compared to the clinically approved PD1 antibodies Pembrolizumab and Nivolumab. Notably, HLX10’s epitope was closer to Pembrolizumab’s epitope than Nivolumab’s epitope. However, HLX10 and Pembrolizumab showed an opposite heavy chain (HC) and light chain (LC) usage, which recognizes several overlapping amino acid residues on PD-1. We compared HLX10 to Nivolumab and Pembrolizumab and it showed similar or better bioactivity in vitro and in vivo, providing a rationale for clinical evaluation in cancer immunotherapy.

Emoxipine Modulates Concentration-Dependent Effects of Cytarabine and Cyclocytidine on Activation of Human T Cells

Title: Emoxipine modulates concentration-dependent effects of cytarabine and cyclocytidine on activation of human T cells. Introduction: Both cytarabine and cyclocytidine are used in the treatment of acute myeloid leukemia. Well known that cytarabine and other related cytosine-based nucleoside analogues are being toxic to tumor cells by increasing levels of cellular oxidative stress as it could be abrogated by antioxidants. However, very little is known both about both the effects of combinations of antimetabolites with antioxidants on the cytotoxic innate and adaptive immune cells and whether lymphocytes toxicity affects its anticancer efficiency. Aim: To estimate effects of cytarabine and cyclocytidine with emoxipine on in vitro activated human T cells at concentrations reached during in vivo treatment with high doses, conventional doses and low doses. Materials and Methods: T cells derived from blood donors were activated in vitro in cell culture medium alone or supplemented with cytarabine 0.1-10.0 μM or cyclocytidine 0.1-10.0 μM. Cell characteristics were assessed by flow cytometry. Results: Only cytarabine 1.0-10.0 μM had both antiproliferative and proapoptotic effects. Additionally, these cytarabine concentrations increased the γIFN-producing by CD3+CD4+ T cells and did not affect the release of this cytokine by CD3+CD8+ T cells. In contrast, the lowest concentration (0.1 μM) did not have or showed minor antiproliferative or cytotoxic effects, did not alter the release of γIFN. Cyclocytidine did not affect viability of normal peripheral blood mononuclear cells but decreased the proliferative capacity of activated normal T cells in dose-dependent manner. Additionally, cyclocytidine altered the percentage of γIFN-producing proliferative CD3+CD8+ cytotoxic T cells for any concentration tested (0.1, 1.0, 1 and 10.0 μM) meanwhile highly suppressed the number of the whole amount of CD3+CD8+ cells and did not affect the release of cytokines by CD3+CD4+ T cells. The study of the expression of the CD107a marker showed a significant stimulating effect of 10 µm of citarabine on the activation of subpopulations of T-lymphocytes (CD3+) and cytotoxic T-lymphocytes (CD3+CD8+).

Discovering tumor-reactive T-cell receptors through single-cell sequencing of tumor-infiltrating lymphocytes

We evaluated the utility of single-cell sequencing of tumor-infiltrating lymphocytes (TIL) for tumor-reactive T-cell receptor (TCR) discovery. Using the MC38 cell line as our tumor model in mice, we show that expression of exogenous TCRs via mRNA electroporation in human T cells provides an easy and quick path to validating tumor-specific candidate TCRs. We detail the identification and validation of four novel MC38-reactive mouse TCRs with varying levels of reactivity to the target cells. Validating our process, one of the MC38 TCRs is specific against a previously reported neoantigen (ASMTNMELM in the Adpgk gene). Consideration of these methodologies may aid in the development of rapid TCR-based therapies for the treatment of cancer and human disease.

Simultaneous assessment of eight phosphorylated STAT residues in T-cells by flow cytometry.

Signal transducer and activator of transcription (STAT) proteins are a family of transcription factors controlling functions in immune responses and other cell types. Given their importance, we developed a flow cytometry panel to assess eight phosphorylated STAT residues in human T-cells, including six tyrosine residues across six STAT proteins (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT6) and additional serine residues on STAT1 and STAT3. We applied this protocol to test the in vitro induction of pSTATs in response to CD3/CD28 activation and a panel of recombinant cytokines. We also assessed the pSTAT expression profiles of naive CD4+ T-cells polarized to Th1, Th2, Th17 or iTregs. pSTAT1(S727), pSTAT2(Y689) and pSTAT3(S727) were constitutively expressed in most T-cells, even in the absence of stimulation. For pSTAT1(S727) and pSTAT3(S727), we observed two positive states, high and low. Conversely, expression of pSTAT1(Y701), pSTAT3(Y705), pSTAT4(Y693) and pSTAT6(Y641) were absent in resting T-cells and only expressed with CD3/CD28 activation or with specific cytokines. Variable frequencies of pSTAT5a(Y694) expression were observed in resting T-cells, which increased with activation or specific cytokine stimulation (e.g. IL-2). IFN-beta stimulation enhanced frequencies of expressing cells for all pSTATs. Correlations among several pSTATs, particularly pSTAT1(S727)high and pSTAT3(S727)high were observed. While polarization resulted in increases in canonically associated pSTATs, other non-canonical pSTAT changes were also observed. Collectively, we developed, optimized, and tested a sensitive and rapid approach for simultaneously assessing phosphorylation of six STAT proteins. Using this approach, we made several novel observations of T-cell pSTAT induction in response to stimuli.

Induction of memory-like CD8 + T cells and CD4 + T cells from human naïve T cells in culture

Memory T cells are crucial players in vertebrate adaptive immunity but their development is incompletely understood. Here we describe a method to produce human memory-like T cells from naïve human T cells in culture. Using commercially available human T cell differentiation kits, both purified naïve CD8 + T cells and purified naïve CD4 + T cells were activated via T cell receptor signaling and appropriate cytokines for several days in culture. All the T cell activators were then removed from the medium and the cultures were continued in hypoxic condition (1% O2 atmosphere) for several more days; during this period, most of the cells died, but some survived in a quiescent state for a month. The survivors had small round cell bodies, expressed differentiation markers characteristic of memory T cells and restarted proliferation when the T cell activators were added back. We could also induce memory-like T cells from naïve human T cells without hypoxia, if we froze the activated T cells or prepared the naïve T cells from chilled filter buffy coats.