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CD28 and CTLA4 are T cell coreceptors that competitively engage B7 ligands CD80 and CD86 to control adaptive immune responses. While the role of CTLA4 in restraining CD28 costimulatory signaling is well-established, the mechanism has remained unclear. Here, we report that human T cells acquire antigen-presenting-cell (APC)–derived B7 ligands and major histocompatibility complex (MHC) via trogocytosis through CD28:B7 binding. Acquired MHC and B7 enabled T cells to autostimulate, and this process was limited cell-intrinsically by CTLA4, which depletes B7 ligands trogocytosed or endogenously expressed by T cells through cis-endocytosis. Extending this model to the previously proposed extrinsic function of CTLA4 in human regulatory T cells (Treg), we show that blockade of either CD28 or CTLA4 attenuates Treg-mediated depletion of APC B7, indicating that trogocytosis and CTLA4-mediated cis-endocytosis work together to deplete B7 from APCs. Our study establishes CTLA4 as a cell-intrinsic molecular sink that limits B7 availability on the surface of T cells, with implications for CTLA4-targeted therapy.

PyMT tumor versus spleen fold change, acute infection day 6 versus spleen fold change.

Selected phosphatases up-regulated in PyMT tumor CD8+ T cells.

PyMT tumor CD8+ versus day 5 effector OT-I gene expression and fold change.

Despite clinical potential and recent advances, durable immunotherapeutic ablation of solid tumors is not routinely achieved. IL15 expands natural killer cell (NK), natural killer T cell (NKT) and CD8+ T-cell numbers and engages the cytotoxic program, and thus is under evaluation for potentiation of cancer immunotherapy. We found that short-term therapy with IL15 bound to soluble IL15 receptor α–Fc (IL15cx; a form of IL15 with increased half-life and activity) was ineffective in the treatment of autochthonous PyMT murine mammary tumors, despite abundant CD8+ T-cell infiltration. Probing of this poor responsiveness revealed that IL15cx only weakly activated intratumoral CD8+ T cells, even though cells in the lung and spleen were activated and dramatically expanded. Tumor-infiltrating CD8+ T cells exhibited cell-extrinsic and cell-intrinsic resistance to IL15. Our data showed that in the case of persistent viral or tumor antigen, single-agent systemic IL15cx treatment primarily expanded antigen-irrelevant or extratumoral CD8+ T cells. We identified exhaustion, tissue-resident memory, and tumor-specific molecules expressed in tumor-infiltrating CD8+ T cells, which may allow therapeutic targeting or programming of specific subsets to evade loss of function and cytokine resistance, and, in turn, increase the efficacy of IL2/15 adjuvant cytokine therapy. Cancer Immunol Res; 4(9); 799–811. ©2016 AACR.

PyMT tumor CD8+ versus day 1 effector OT-I gene expression and fold change.

Supplemental Figure 1: Five day IL-15cx treatment results in splenic CD8+ T cell activation and up-regulation of cytotoxic molecule expression in PyMT tumor-bearing mice. Supplemental Figure 2: PyMT tumor single-cell suspensions induce IL-2 / IL-15 resistance in CD8+ T cells. Supplemental Figure 3: Abundance of IL-2 / IL-15 cytokine receptor chains in PyMT tumor, acute/persistent infection, and TRM CD8+ T cells. Supplemental Figure 4: Gene expression in tumor versus acute infection CD8+ T cells. Supplemental Figure 5: Differentially-regulated transcripts in PyMT tumor CD8+ T cells versus naive, early effector, peak effector and memory T cells. Supplemental Figure 6: Human breast cancer CD8+ T cell infiltrate. Supplemental Figure 7: TRM "core signature" gene expression and transcriptional regulator TOX correlation with IL-15 resistance. Supplemental Figure 8: IL-15cx treatment of persistent virus-infected mice results in decreased relative abundance of exhausted antigen-specific CD8+ T cells. Supplemental Figure 9: Combined in vivo IL-15cx and αPD-L1 antibody treatment does not rescue PyMT CD8+ T cell cytokine non-responsiveness. Supplemental Figure 10: Expression of CD80 and CD200R on CD8+ T cells from PyMT tumor and persistent viral infection. Supplemental Figure 11: Activated and expanded tumor-specific CD8+ T cells adoptively transferred to tumor-bearing mice localize to tumor, but rapidly up-regulate PD-1 and CD244.

Transcripts up-regulated {greater than or equal to}2-fold in PyMT tumor versus splenic CD8+ T cells.

PyMT tumor CD8+ versus naive gene expression and fold change.

Supplementary Data from AMC-070: Lenalidomide Is Safe and Effective in HIV-Associated Kaposi Sarcoma

Purpose:Kaposi sarcoma (KS), an endothelial cell tumor associated with KS herpesvirus (KSHV), remains among the most common malignancies occurring with HIV infection (HIV-KS). As an oral anti-inflammatory, antiangiogenic, and immunomodulatory agent, lenalidomide is potentially an attractive alternative to standard chemotherapy for KS.Experimental Design:The primary objectives of this phase I/II trial were to determine the maximum tolerated dose (MTD) and response rates for lenalidomide in HIV-KS. Secondary objectives included correlating response with natural killer (NK) and T-cell subsets, plasma cytokines, viral copy number, and KSHV gene expression in biopsies. Four dose levels of oral lenalidomide taken 21 consecutive days of 28-day cycles were evaluated in adults with HIV-KS on antiretroviral therapy with controlled viremia.Results:Fifteen and 23 participants enrolled in phases I and II, respectively, 76% of whom had received prior KS therapy. The MTD was not reached, declaring 25 mg as the recommended phase II dose (RP2D). The most frequent adverse events were neutropenia, fatigue, leukopenia, and diarrhea. Of the 25 evaluable participants receiving RP2D, 60% responded. Correlative studies performed in a subset of participants demonstrated a significant increase in proportions of blood T cells with T-regulatory phenotype, and plasma cytokines trended toward a less inflammatory pattern. Clinical response was associated with loss of KSHV transcription.Conclusions:Lenalidomide is active in HIV-KS. The most common adverse events were manageable. With 60% of participants receiving RP2D obtaining a partial response and See related commentary by Henry and Maki, p. 2485

Expression of the TAM (TYRO3, AXL, MER) family of receptor tyrosine kinases (RTK) has been associated with cancer progression, metastasis, and drug resistance. In immune cells, TAM RTKs can dampen inflammation in favor of homeostatic wound-healing responses, thus potentially contributing to the evasion of cancer cells from immune surveillance. Here we characterize the small-molecule RXDX-106 as a selective and potent pan-TAM RTK inhibitor with slow dissociation kinetics and significant antitumor activity in multiple syngeneic tumor models. Expression of AXL and MER on both immune and tumor cells increased during tumor progression. Tumor growth inhibition (TGI) following treatment with RXDX-106 was observed in wild-type mice and was abrogated in immunodeficient mice, suggesting that the antitumor activity of RXDX-106 is, in part, due to the presence of immune cells. RXDX-106–mediated TGI was associated with increased tumor-infiltrating leukocytes, M1-polarized intratumoral macrophages, and activation of natural killer cells. RXDX-106 proportionally increased intratumoral CD8+ T cells and T-cell function as indicated by both IFNγ production and LCK phosphorylation (pY393). RXDX-106 exhibited its effects via direct actions on TAM RTKs expressed on intratumoral macrophages and dendritic cells, leading to indirect activation of other immune cells in the tumor. RXDX-106 also potentiated the effects of an immune checkpoint inhibitor, α-PD-1 Ab, resulting in enhanced antitumor efficacy and survival. Collectively, these results demonstrate the capacity of RXDX-106 to inhibit tumor growth and progression and suggest it may serve as an effective therapy against multiple tumor types.Significance:The pan-TAM small-molecule kinase inhibitor RXDX-106 activates both innate and adaptive immunity to inhibit tumor growth and progression, indicating its clinical potential to treat a wide variety of cancers.

Listed are the Top 100 genes significantly upregulated or downregulated over time in the MC38 model by RXDX-106 treatment relative to vehicle.

PDF file - 25K, Cytotoxicity of neutrophils and monocytes/macrophages

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PDF file - 21K, Strain-specific differences in Yac-1 cytotoxcity

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PDF file - 76K, Microsatellite mapping of the 129/SvEv.B6-NKC Rag2-/- mouse

PDF file - 30K, Cytotoxicity of NK cells from C57BL/10 and Balb/c mice

PDF file - 36K, T cell depletion in AJ mice

PDF file - 47K, SNP rs13459098 in the Pas-1 locus is of 129 origin in 129/SvEv.B6-NKC Rag2-/- mice

Purpose:Kaposi sarcoma (KS), an endothelial cell tumor associated with KS herpesvirus (KSHV), remains among the most common malignancies occurring with HIV infection (HIV-KS). As an oral anti-inflammatory, antiangiogenic, and immunomodulatory agent, lenalidomide is potentially an attractive alternative to standard chemotherapy for KS.Experimental Design:The primary objectives of this phase I/II trial were to determine the maximum tolerated dose (MTD) and response rates for lenalidomide in HIV-KS. Secondary objectives included correlating response with natural killer (NK) and T-cell subsets, plasma cytokines, viral copy number, and KSHV gene expression in biopsies. Four dose levels of oral lenalidomide taken 21 consecutive days of 28-day cycles were evaluated in adults with HIV-KS on antiretroviral therapy with controlled viremia.Results:Fifteen and 23 participants enrolled in phases I and II, respectively, 76% of whom had received prior KS therapy. The MTD was not reached, declaring 25 mg as the recommended phase II dose (RP2D). The most frequent adverse events were neutropenia, fatigue, leukopenia, and diarrhea. Of the 25 evaluable participants receiving RP2D, 60% responded. Correlative studies performed in a subset of participants demonstrated a significant increase in proportions of blood T cells with T-regulatory phenotype, and plasma cytokines trended toward a less inflammatory pattern. Clinical response was associated with loss of KSHV transcription.Conclusions:Lenalidomide is active in HIV-KS. The most common adverse events were manageable. With 60% of participants receiving RP2D obtaining a partial response and See related commentary by Henry and Maki, p. 2485

In the tumor microenvironment, immune cells and tumor cells interact in a process called cancer immunoediting, giving rise to changes in gene expression, metabolism, mutational burden, and cellularity in the tumor. This SnapShot compares endogenous versus therapy-induced cancer immunoediting and outlines the molecular and cellular characteristics of interactions that result in complete tumor regression versus tumor escape and progression. To view this SnapShot, open or download the PDF.

BackgroundDiscovery that ~16% of T cells naturally co-express two T-cell receptor (TCR) clonotypes prompts examining the role of dual TCR cells in immune functions.MethodsUsing TCRα-reporter transgenic mice, enabling unambiguous identification of single-TCR and dual-TCR cells, we tested the role of dual TCR cells in antitumor immune responses against immune-responsive syngeneic 6727 sarcoma and immune-resistant B16F10 melanoma.ResultsDual TCR cells were specifically increased among tumor-infiltrating lymphocytes (TILs) in both models, indicating selective advantage in antitumor responses. Phenotype and single-cell gene expression analyses identified dual TCR are predominant during the effective antitumor response, demonstrating selectively increased activation in the TIL compartment and skewing toward an effector memory phenotype. Absence of dual TCR cells impaired immune response to B16F10 but not 6727, suggesting that dual TCR cells may be more influential in responses against poorly immunogenic tumors. Dual TCR cells demonstrated an advantage in recognition of B16F10-derived neoantigens in vitro, providing a mechanistic basis for their antitumor reactivity.ConclusionsThese results discover an unrecognized role for dual TCR cells in protective immune function and identify these cells and their TCRs as a potential resource for antitumor immunotherapy.

Epidemiological studies demonstrate an association between breast cancer (BC) and systemic dysregulation of glucose metabolism. However, how BC influences glucose homeostasis remains unknown. We show that BC-derived extracellular vesicles (EVs) suppress pancreatic insulin secretion to impair glucose homeostasis. EV-encapsulated miR-122 targets PKM in β-cells to suppress glycolysis and ATP-dependent insulin exocytosis. Mice receiving high-miR-122 EVs or bearing BC tumours exhibit suppressed insulin secretion, enhanced endogenous glucose production, impaired glucose tolerance and fasting hyperglycaemia. These effects contribute to tumour growth and are abolished by inhibiting EV secretion or miR-122, restoring PKM in β-cells or supplementing insulin. Compared with non-cancer controls, patients with BC have higher levels of circulating EV-encapsulated miR-122 and fasting glucose concentrations but lower fasting insulin; miR-122 levels are positively associated with glucose and negatively associated with insulin. Therefore, EV-mediated impairment of whole-body glycaemic control may contribute to tumour progression and incidence of type 2 diabetes in some patients with BC.

Absence of adequate CD28 costimulation is a common feature of dysfunctional T cells in cancer. However, although augmentation of CD28 signaling is well known to be essential for restoring dysfunctional T cell immune responses upon immune checkpoint blockade treatment, it is unclear how CD28 in tumor-infiltrated T cells is activated in the absence of corresponding ligands on cancer. In current dogma, the T cell CD28 can be activated in trans by CD80 (B7-1) or CD86 (B7-2) expressed on professional antigen presenting cells (APCs) enriched in secondary lymphoid organs. Notably, besides professional APCs, B7 ligands are also displayed by T cells, but their functions are unclear. Here we report that B7 ligands expressed on T cells interact with CD28 in cis at membrane invaginations of the immunological synapse, as a result of phosphoinositide-3-kinase (PI3K) mediated membrane remodeling. Cis-B7:CD28 interactions triggered CD28 signaling through protein-kinase-C-theta (PKCθ) and promoted T cell survival, migration and cytokine production. In a B7 deficient tumor model grafted with primed CD8+ T cells, blockade of T cell intrinsic B7:CD28 interactions accelerated tumor growth and decreased intratumoral T cells. Thus, B7 ligands on T cells can evoke cell autonomous CD28 costimulation in cis in peripheral tissues, suggesting cis-signaling as a general mechanism for boosting T cell functionality. Citation Format: Yunlong Zhao, Christine Caron, Ya-Yuan Chan, Calvin K. Lee, Xiaozheng Xu, Jack D. Bui, Enfu Hui. Cis-B7:CD28 interactions at invaginated synaptic membranes activate CD28 and promote T cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 606.

IL-17D is a cytokine that belongs to the IL-17 family and is conserved in vertebrates and invertebrates. In contrast to IL-17A and IL-17F, which are expressed in Th17 cells, IL-17D is expressed broadly in nonimmune cells. IL-17D can promote immune responses to cancer and viruses in part by inducing chemokines and recruiting innate immune cells such as NK cells. Although bacterial infection can induce IL-17D in fish and invertebrates, the role of mammalian IL-17D in antibacterial immunity has not been established. To determine whether IL-17D has a role in mediating host defense against bacterial infections, we studied i.p. infection by group A Streptococcus (GAS) in wild-type (WT) and Il17d−/− mice. Compared with WT animals, mice deficient in IL-17D experienced decreased survival, had greater weight loss, and showed increased bacterial burden in the kidney and peritoneal cavity following GAS challenge. In WT animals, IL-17D transcript was induced by GAS infection and correlated to increased levels of chemokine CCL2 and greater neutrophil recruitment. Of note, GAS-mediated IL-17D induction in nonimmune cells required live bacteria, suggesting that processes beyond recognition of pathogen-associated molecular patterns were required for IL-17D induction. Based on our results, we propose a model in which nonimmune cells can discriminate between nonviable and viable GAS cells, responding only to the latter by inducing IL-17D.

Summary Background Refractory coeliac disease type 2 is a rare subtype of coeliac disease with high mortality rates; interleukin 15 (IL-15) is strongly implicated in its pathophysiology. This trial aimed to investigate the effects of AMG 714, an anti-IL-15 monoclonal antibody, on the activity and symptoms of refractory coeliac disease type 2. Methods This was a randomised, double-blind, placebo-controlled, phase 2a study of adults with a confirmed diagnosis of refractory coeliac disease type 2. Patients were randomly assigned at a 2:1 ratio to receive seven intravenous doses over 10 weeks of AMG 714 (8 mg/kg) or matching placebo. Biopsy samples were obtained at baseline and week 12 for cellular analysis and histology. The change in the proportion of aberrant intraepithelial lymphocytes from baseline to week 12 with respect to all intraepithelial lymphocytes was the primary endpoint and was quantified using flow cytometry. Secondary endpoints were the change in aberrant intraepithelial lymphocytes with respect to intestinal epithelial cells; intestinal histological scores (villous height-to-crypt depth ratio; VHCD); intraepithelial lymphocyte counts; Marsh score; and patient-reported symptom measures, including the Bristol stool form scale (BSFS) and gastrointestinal symptom rating scale (GSRS). Main analyses were done in the per-protocol population of patients who received their assigned treatment, provided evaluable biopsy samples, and did not have major protocol deviations; only patients with non-atypical disease were included in the analyses of aberrant intraepithelial lymphocytes, including the primary analysis. Safety was assessed in all patients who received at least one dose of study drug. This study is registered at ClinicalTrials.gov (NCT02633020) and EudraCT (2015-004063-36). Findings From April 13, 2016, to Jan 19, 2017, 28 patients were enrolled and randomly assigned to AMG 714 (n=19) and placebo (n=9). Six patients were not included in the primary analysis because of protocol deviation (one in the AMG 714 group), insufficient biopsy samples (one in the AMG 714 group), and atypical intraepithelial lymphocytes (three in the AMG 714 group and one in the placebo group). At 12 weeks, the least square mean difference between AMG 714 and placebo in the relative change from baseline in aberrant intraepithelial lymphocyte percentage was −4·85% (90% CI −30·26 to 20·56; p=0·75). The difference between the AMG 714 and placebo groups in aberrant intraepithelial lymphocytes with respect to epithelial cells at 12 weeks was −38·22% (90% CI −95·73 to 19·29; nominal p=0·18); the difference in change in Marsh score from baseline was 0·09% (95% CI −1·60–1·90; nominal p=0·92); the difference in VHCD ratio was 10·67% (95% CI −38·97 to 60·31; nominal p=0·66); and the difference in change in total intraepithelial lymphocyte count was −12·73% (95% CI −77·57–52·12); nominal p=0·69). Regarding symptoms, the proportion of patients with diarrhoea per the BSFS score decreased from ten (53%) of 19 at baseline to seven (37%) of 19 at week 12 in the AMG 714 group and increased from two (22%) of nine at baseline to four (44%) of nine at week 12 in the placebo group (nominal p=0·0008); and the difference between the groups in change in GSRS score was −0·14 (SE 0·19; nominal p=0·48). Eight (89%) patients in the placebo group and 17 (89%) in the AMG 714 group had treatment-emergent adverse events, including one (11%) patient in the placebo group and five (26%) in the AMG 714 group who had serious adverse events. The most common adverse event in the AMG 714 group was nasopharyngitis (eight [42%] patients vs one [11%] in the placebo group). Interpretation In patients with refractory coeliac disease type 2 who were treated with AMG 714 or placebo for 10 weeks, there was no difference between the groups in terms of the primary endpoint of aberrant intraepithelial lymphocyte reduction from baseline. Effects on symptoms and other endpoints suggest that further research of AMG 714 may be warranted in patients with refractory coeliac disease type 2. Funding Celimmune and Amgen.

High‐throughput single‐cell cytometry technologies have significantly improved our understanding of cellular phenotypes to support translational research and the clinical diagnosis of hematological and immunological diseases. However, subjective and ad hoc manual gating analysis does not adequately handle the increasing volume and heterogeneity of cytometry data for optimal diagnosis. Prior work has shown that machine learning can be applied to classify cytometry samples effectively. However, many of the machine learning classification results are either difficult to interpret without using characteristics of cell populations to make the classification, or suboptimal due to the use of inaccurate cell population characteristics derived from gating boundaries. To date, little has been done to optimize both the gating boundaries and the diagnostic accuracy simultaneously. In this work, we describe a fully discriminative machine learning approach that can simultaneously learn feature representations (e.g., combinations of coordinates of gating boundaries) and classifier parameters for optimizing clinical diagnosis from cytometry measurements. The approach starts from an initial gating position and then refines the position of the gating boundaries by gradient descent until a set of globally‐optimized gates across different samples are achieved. The learning procedure is constrained by regularization terms encoding domain knowledge that encourage the algorithm to seek interpretable results. We evaluate the proposed approach using both simulated and real data, producing classification results on par with those generated via human expertise, in terms of both the positions of the gating boundaries and the diagnostic accuracy. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

The molecular underpinnings of acquired resistance to carboplatin are poorly understood and often inconsistent between in vitro modeling studies. After sequential treatment cycles, multiple isogenic clones reached similar levels of resistance, but significant transcriptional heterogeneity. Gene-expression based virtual synchronization of 26,772 single cells from 2 treatment steps and 4 resistant clones was used to evaluate the activity of Hallmark gene sets in proliferative (P) and quiescent (Q) phases. Two behaviors were associated with resistance: (1) broad repression in the P phase observed in all clones in early resistant steps and (2) prevalent induction in Q phase observed in the late treatment step of one clone. Furthermore, the induction of IFNα response in P phase or Wnt-signaling in Q phase were observed in distinct resistant clones. These observations suggest a model of resistance hysteresis, where functional alterations of the P and Q phase states affect the dynamics of the successive transitions between drug exposure and recovery, and prompts for a precise monitoring of single-cell states to develop more effective schedules for, or combination of, chemotherapy treatments.

Locally advanced cancers remain therapeutically challenging to eradicate. The most successful treatments continue to combine decades old non-targeted chemotherapies with radiotherapy that unfortunately increase normal tissue damage in the irradiated field and have systemic toxicities precluding further treatment intensification. Therefore, alternative molecularly guided systemic therapies are needed to improve patient outcomes when applied with radiotherapy. In this work, we report a trimodal precision cytotoxic chemo-radio-immunotherapy paradigm using spatially targeted auristatin warheads. Tumor-directed antibodies and peptides conjugated to radiosensitizing monomethyl auristatin E (MMAE) specifically produce CD8 T cell dependent durable tumor control of irradiated tumors and immunologic memory. In combination with ionizing radiation, MMAE sculpts the tumor immune infiltrate to potentiate immune checkpoint inhibition. Here, we report therapeutic synergies of targeted cytotoxic auristatin radiosensitization to stimulate anti-tumor immune responses providing a rationale for clinical translational of auristatin antibody drug conjugates with radio-immunotherapy combinations to improve tumor control.

Expression of the TAM (TYRO3, AXL, MER) family of receptor tyrosine kinases (RTK) has been associated with cancer progression, metastasis, and drug resistance. In immune cells, TAM RTKs can dampen inflammation in favor of homeostatic wound-healing responses, thus potentially contributing to the evasion of cancer cells from immune surveillance. Here we characterize the small-molecule RXDX-106 as a selective and potent pan-TAM RTK inhibitor with slow dissociation kinetics and significant antitumor activity in multiple syngeneic tumor models. Expression of AXL and MER on both immune and tumor cells increased during tumor progression. Tumor growth inhibition (TGI) following treatment with RXDX-106 was observed in wild-type mice and was abrogated in immunodeficient mice, suggesting that the antitumor activity of RXDX-106 is, in part, due to the presence of immune cells. RXDX-106–mediated TGI was associated with increased tumor-infiltrating leukocytes, M1-polarized intratumoral macrophages, and activation of natural killer cells. RXDX-106 proportionally increased intratumoral CD8+ T cells and T-cell function as indicated by both IFNγ production and LCK phosphorylation (pY393). RXDX-106 exhibited its effects via direct actions on TAM RTKs expressed on intratumoral macrophages and dendritic cells, leading to indirect activation of other immune cells in the tumor. RXDX-106 also potentiated the effects of an immune checkpoint inhibitor, α-PD-1 Ab, resulting in enhanced antitumor efficacy and survival. Collectively, these results demonstrate the capacity of RXDX-106 to inhibit tumor growth and progression and suggest it may serve as an effective therapy against multiple tumor types. Significance: The pan-TAM small-molecule kinase inhibitor RXDX-106 activates both innate and adaptive immunity to inhibit tumor growth and progression, indicating its clinical potential to treat a wide variety of cancers.

Intratumoral immunotherapy is an emerging modality for the treatment of solid tumors. Toll-like receptor (TLR) agonists have shown promise for eliciting immune responses, but systemic administration often results in the development of adverse side effects. Herein, we investigate whether localized delivery of the TLR agonist, resiquimod (R848), via platelet membrane-coated nanoparticles (PNP-R848) elicits antitumor responses. The membrane coating provides a means of enhancing interactions with the tumor microenvironment, thereby maximizing the activity of R848. Intratumoral administration of PNP-R848 strongly enhances local immune activation and leads to complete tumor regression in a colorectal tumor model, while providing protection against repeated tumor re-challenges. Moreover, treatment of an aggressive breast cancer model with intratumoral PNP-R848 delays tumor growth and inhibits lung metastasis. Our findings highlight the promise of locally delivering immunostimulatory payloads using biomimetic nanocarriers, which possess advantages such as enhanced biocompatibility and natural targeting affinities., The immunostimulatory properties of TLR7/8 agonists, such as resiquimod, have been exploited for cancer immunotherapy. Here, the authors design platelet membrane-cloaked nanoparticles for selective intratumoral delivery of resiquimod, resulting in potent anti-tumor immune response in a range of preclinical solid tumors.

During bacterial sepsis, tethers break off from neutrophils rolling on the vessel wall, destabilizing neutrophil rolling and forming elongated neutrophil-derived structures (ENDS) in the blood circulation. ENDS are phosphatidylserine- and tetraspanin-negative structures that fragment, round up, and release S100-A8/A9 upon degradation., Rolling neutrophils form tethers with submicron diameters. Here, we report that these tethers detach, forming elongated neutrophil-derived structures (ENDS) in the vessel lumen. We studied ENDS formation in mice and humans in vitro and in vivo. ENDS do not contain mitochondria, endoplasmic reticulum, or DNA, but are enriched for S100A8, S100A9, and 57 other proteins. Within hours of formation, ENDS round up, and some of them begin to present phosphatidylserine on their surface (detected by annexin-5 binding) and release S100A8–S100A9 complex, a damage-associated molecular pattern protein that is a known biomarker of neutrophilic inflammation. ENDS appear in blood plasma of mice upon induction of septic shock. Compared with healthy donors, ENDS are 10–100-fold elevated in blood plasma of septic patients. Unlike neutrophil-derived extracellular vesicles, most ENDS are negative for the tetraspanins CD9, CD63, and CD81. We conclude that ENDS are a new class of bloodborne submicron particles with a formation mechanism linked to neutrophil rolling on the vessel wall., Graphical Abstract

Natural killer (NK) cells are innate immune lymphocytes that recognize and destroy abnormal host cells, such as tumor cells or those infected by viral pathogens. To safely accomplish these functions, NK cells display activating receptors that detect stress molecules or viral ligands displayed at the cell surface, balanced by inhibitory receptors that bind to self-molecules. To date, such activating and inhibitory receptors on NK cells are not known to recognize bacterial determinants. Moreover, NK cell responses to direct interactions with extracellular bacteria are poorly explored. In this study, we observed the human neonatal pathogen group B Streptococcus (GBS) can directly engage human NK cells. The interaction was mediated through the B6N segment of streptococcal β-protein, binding to the inhibitory receptor Siglec-7 via its amino-terminal V-set domain. Unlike classical Siglec binding, the interaction is also independent of its sialic acid recognition property. In contrast to WT GBS, mutants lacking β-protein induced efficient pyroptosis of NK cells through the NLRP3 inflammasome, with production and secretion of the proinflammatory cytokine IL-1β and dissemination of the cytotoxic molecule granzyme B. We postulate that GBS evolved β-protein engagement of inhibitory human Siglec-7 to suppress the pyroptotic response of NK cells and thereby block recruitment of a broader innate immune response, i.e., by "silencing the sentinel."

Interleukin-17D (IL-17D) is a cytokine that belongs to the IL-17 family and is conserved in vertebrates and invertebrates. In contrast to IL-17A and IL-17F, which are expressed in Th-17 cells, IL-17D is expressed broadly in non-immune cells. IL-17D can promote immune responses to cancer and viruses in part by inducing chemokines and recruiting innate immune cells such as natural killer (NK) cells. Although bacterial infection can induce IL-17D in fish and invertebrates, the role of mammalian IL-17D in anti-bacterial immunity has not been established. To determine whether IL-17D has a role in mediating host defense against bacterial infections, we studied intraperitoneal infection by group A Streptococcus (GAS) in wild-type (WT) and Il17d(−/−) mice. Compared to WT animals, mice deficient in IL-17D experienced decreased survival, had greater weight loss, and showed increased bacterial burden in the kidney and peritoneal cavity following GAS challenge. In WT animals, IL-17D transcript was induced by GAS infection and correlated to increased levels of chemokine CCL2 and greater neutrophil recruitment. Of note, GAS-mediated IL-17D induction in non-immune cells required live bacteria, suggesting that processes beyond recognition of pathogen-associated molecular patterns were required for IL-17D induction. Based on our results, we propose a model in which non-immune cells can discriminate between non-viable and viable GAS cells, responding only to the latter by inducing IL-17D.

Combined immunotherapy targeting the immune checkpoint receptors cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1), or CTLA-4 and the PD-1 ligand (PD-L1) exhibits superior anti-tumor responses compared with single-agent therapy. Here, we examined the molecular basis for this synergy. Using reconstitution assays with fluorescence readouts, we found that PD-L1 and the CTLA-4 ligand CD80 heterodimerize in cis but not trans. Quantitative biochemistry and cell biology assays revealed that PD-L1:CD80 cis-heterodimerization inhibited both PD-L1:PD-1 and CD80:CTLA-4 interactions through distinct mechanisms but preserved the ability of CD80 to activate the Tcell co-stimulatory receptor CD28. Furthermore, PD-L1 expression on antigen-presenting cells (APCs) prevented CTLA-4-mediated trans-endocytosis of CD80. Atezolizumab (anti-PD-L1), but not anti-PD-1, reduced cell surface expression of CD80 on APCs, and this effect was negated by co-blockade of CTLA-4 with ipilimumab (anti-CTLA-4). Thus, PD-L1 exerts an immunostimulatory effect by repressing the CTLA-4 axis; this has implications to the synergy of anti-PD-L1 and anti-CTLA-4 combination therapy.

Cell-cell fusion describes the process by which two cells combine their plasma membranes and become a single cell, possessing and retaining certain genetic information from each parent cell. Here, using a Cre-loxP-based method initially developed to investigate extracellular vesicle targeting, we found that cancer cells spontaneously and rapidly deliver DNA to non-cancer cells in vitro via a cell-cell fusion event. The resulting hybrid cells were aneuploid and possessed enhanced clonal diversity and chemoresistance compared to non-hybrid cancer cells. We also observed cell-cell fusion to occur in vivo between melanoma cells and non-cancer cells of both hematopoietic and non-hematopoietic lineages. These findings suggest that cell-cell fusion occurs during the natural progression of cancer and show that this mechanism has the potential to cause massive genomic alterations that are observed in cancer. Furthermore, these findings somewhat contradict recent publications suggesting that the Cre-loxP method measures only extracellular vesicle-mediated intercellular communication.

Flow cytometry is used in cell-based diagnostic evaluation for blood-borne malignancies including leukemia and lymphoma. The current practice for cytometry data analysis relies on manual gating to identify cell subsets in complex mixtures, which is subjective, labor-intensive, and poorly reproducible. This article reviews recent efforts to develop, validate, and disseminate automated computational methods and pipelines for cytometry data analysis that could help overcome the limitations of manual analysis and provide for efficient and data-driven diagnostic applications. It demonstrates the performance of an optimized computational pipeline in a pilot study of chronic lymphocytic leukemia data from the authors' clinical diagnostic laboratory.

The purpose of this review is to explore immune-mediated mechanisms of stress surveillance in cancer, with particular emphasis on the idea that all cancers have classical hallmarks (Hanahan and Weinberg in Cell 100:57-70, 67; Cell 144:646-674, 68) that could be interrelated. We postulate that hallmarks of cancer associated with cellular stress pathways (Luo et al. in Cell 136:823-837, 101) including oxidative stress, proteotoxic stress, mitotic stress, DNA damage, and metabolic stress could define and modulate the inflammatory component of cancer. As such, the overarching goal of this review is to define the types of cellular stress that cancer cells undergo, and then to explore mechanisms by which immune cells recognize, respond to, and are affected by each stress response.