Improving radiotherapy in immunosuppressive microenvironments by targeting complement receptor C5aR1.

An immunosuppressive microenvironment causes poor tumor T cell infiltration and is associated with reduced patient overall survival in colorectal cancer. How to improve treatment responses in these tumors is still a challenge. Using an integrated screening approach to identify cancer-specific vulnerabilities, we identified complement receptor C5aR1 as a druggable target, which when inhibited improved radiotherapy, even in tumors displaying immunosuppressive features and poor CD8+ T cell infiltration. While C5aR1 is well-known for its role in the immune compartment, we found that C5aR1 is also robustly expressed on malignant epithelial cells, highlighting potential tumor cell-specific functions. C5aR1 targeting resulted in increased NF-κB-dependent apoptosis specifically in tumors and not normal tissues, indicating that, in malignant cells, C5aR1 primarily regulated cell fate. Collectively, these data revealed that increased complement gene expression is part of the stress response mounted by irradiated tumors and that targeting C5aR1 could improve radiotherapy, even in tumors displaying immunosuppressive features.

Mass cytometric analysis of the immune cell landscape after traumatic brain injury elucidates the role of complement and complement receptors in neurologic outcomes.

Following traumatic brain injury (TBI), a neuroinflammatory response can persist for years and contribute to the development of chronic neurological manifestations. Complement plays a central role in post-TBI neuroinflammation, and C3 opsonins and the anaphylatoxins (C3a and C5a) have been implicated in promoting secondary injury. We used single cell mass cytometry to characterize the immune cell landscape of the brain at different time points after TBI. To specifically investigate how complement shapes the post-TBI immune cell landscape, we analyzed TBI brains in the context of CR2-Crry treatment, an inhibitor of C3 activation. We analyzed 13 immune cell types, including peripheral and brain resident cells, and assessed expression of various receptors. TBI modulated the expression of phagocytic and complement receptors on both brain resident and infiltrating peripheral immune cells, and distinct functional clusters were identified within same cell populations that emerge at different phases after TBI. In particular, a CD11c+ (CR4) microglia subpopulation continued to expand over 28 days after injury, and was the only receptor to show continuous increase over time. Complement inhibition affected the abundance of brain resident immune cells in the injured hemisphere and impacted the expression of functional receptors on infiltrating cells. A role for C5a has also been indicated in models of brain injury, and we found significant upregulation of C5aR1 on many immune cell types after TBI. However, we demonstrated experimentally that while C5aR1 is involved in the infiltration of peripheral immune cells into the brain after injury, it does not alone affect histological or behavioral outcomes. However, CR2-Crry improved post-TBI outcomes and reduced resident immune cell populations, as well as complement and phagocytic receptor expression, indicating that its neuroprotective effects are mediated upstream of C5a generation, likely via modulating C3 opsonization and complement receptor expression.

A role for P-selectin and complement in the pathological sequelae of germinal matrix hemorrhage.

BACKGROUND: Germinal matrix hemorrhage is a devastating disease of pre-term infancy commonly resulting in post-hemorrhagic hydrocephalus, periventricular leukomalacia, and subsequent neurocognitive deficits. We demonstrate vascular expression of the adhesion molecule P-selectin after GMH and investigate a strategy to specifically target complement inhibition to sites of P-selectin expression to mitigate the pathological sequelae of GMH. METHODS: We prepared two fusion proteins consisting of different anti-P-selectin single chain antibodies (scFv's) linked to the complement inhibitor Crry. One scFv targeting vehicle (2.12scFv) blocked the binding of P-selectin to its PSGL-1 ligand expressed on leukocytes, whereas the other targeting vehicle (2.3scFv) bound P-selectin without blocking ligand binding. Post-natal C57BL/6 J mice on day 4 (P4) were subjected to collagenase induced-intraventricular hemorrhage and treated with 2.3Psel-Crry, 2.12Psel-Crry, or vehicle. RESULTS: Compared to vehicle treatment, 2.3Psel-Crry treatment after induction of GMH resulted in reduced lesion size and mortality, reduced hydrocephalus development, and improved neurological deficit measurements in adolescence. In contrast, 2.12Psel-Crry treatment resulted in worse outcomes compared to vehicle. Improved outcomes with 2.3Psel-Crry were accompanied by decreased P-selectin expression, and decreased complement activation and microgliosis. Microglia from 2.3Psel-Crry treated mice displayed a ramified morphology, similar to naïve mice, whereas microglia in vehicle treated animals displayed a more ameboid morphology that is associated with a more activated status. Consistent with these morphological characteristics, there was increased microglial internalization of complement deposits in vehicle compared to 2.3Psel-Crry treated animals, reminiscent of aberrant C3-dependent microglial phagocytosis that occurs in other (adult) types of brain injury. In addition, following systemic injection, 2.3Psel-Crry specifically targeted to the post-GMH brain. Likely accounting for the unexpected finding that 2.12Psel-Crry worsens outcome following GMH was the finding that this construct interfered with coagulation in this hemorrhagic condition, and specifically with heterotypic platelet-leukocyte aggregation, which express P-selectin and PSGL-1, respectively. CONCLUSIONS: GMH induces expression of P-selectin, the targeting of which with a complement inhibitor protects against pathogenic sequelae of GMH. A dual functioning construct with both P-selectin and complement blocking activity interferes with coagulation and worsens outcomes following GMH, but has potential for treatment of conditions that incorporate pathological thrombotic events, such as ischemic stroke.

A Role for P-selectin and Complement in the Pathological Sequelae of Germinal Matrix Hemorrhage.

Background Germinal Matrix Hemorrhage is a devastating disease of pre-term infancy commonly resulting in post-hemorrhagic hydrocephalus, periventricular leukomalacia, and subsequent neurocognitive deficits. We demonstrate vascular expression of the adhesion molecule P-selectin after GMH and investigate a strategy to specifically target complement inhibition to sites of P-selectin expression to mitigate the pathological sequelae of GMH. Methods We prepared two fusion proteins consisting of different anti-P-selectin single chain antibodies (scFv's) linked to the complement inhibitor Crry. One scFv targeting vehicle (2.12scFv) blocked the binding of P-selectin to its PSGL-1 ligand expressed on leukocytes, whereas the other targeting vehicle (2.3scFv) bound P-selectin without blocking ligand binding. Post-natal mice on day 4 (P4) were subjected to collagenase induced-intraventricular hemorrhage and treated with 2.3Psel-Crry, 2.12Psel-Crry, or vehicle. Results Compared to vehicle treatment, 2.3Psel-Crry treatment after induction of GMH resulted in reduced lesion size and mortality, reduced hydrocephalus development, and improved neurological deficit measurements in adolescence. In contrast, 2.12Psel-Crry treatment resulted in worse outcomes compared to vehicle. Improved outcomes with 2.3Psel-Crry were accompanied by decreased P-selectin expression, and decreased complement activation and microgliosis. Microglia from 2.3Psel-Crry treated mice displayed a ramified morphology, similar to naïve mice, whereas microglia in vehicle treated animals displayed a more ameboid morphology that is associated with a more activated status. Consistent with these morphological characteristics, there was increased microglial internalization of complement deposits in vehicle compared to 2.3Psel-Crry treated animals, reminiscent of aberrant C3-dependent microglial phagocytosis that occurs in other (adult) types of brain injury. Also, following systemic injection, 2.3Psel-Crry specifically targeted to the post-GMH brain. Likely accounting for the unexpected finding that 2.12Psel-Crry worsens outcome following GMH was the finding that this construct interfered with coagulation in this hemorrhagic condition, and specifically with heterotypic platelet-leukocyte aggregation, which express P-selectin and PSGL-1, respectively. Conclusion GMH induces expression of P-selectin, the targeting of which with a complement inhibitor protects against pathogenic sequelae of GMH. A dual functioning construct with both P-selectin and complement blocking activity interferes with coagulation and worsens outcomes following GMH, but has potential for treatment of conditions that incorporate pathological thrombotic events, such as ischemic stroke.

Phenotypic and functional heterogeneity of monocytes in health and cancer in the era of high dimensional technologies.

Monocytes have been traditionally classified in three discrete subsets, which can participate in the immune responses as effector cells or as precursors of myeloid-derived cells in circulation and tissues. However, recent advances in single-cell omics have revealed unprecedented phenotypic and functional heterogeneity that goes well beyond the three conventional monocytic subsets and propose a more fluid differentiation model. This novel concept does not only apply to the monocytes in circulation but also at the tissue site. Consequently, the binary model proposed for differentiating monocyte into M1 and M2 macrophages has been recently challenged by a spectrum model that more realistically mirrors the heterogeneous cues in inflammatory conditions. This review describes the latest results on the high dimensional characterization of monocytes and monocyte-derived myeloid cells in steady state and cancer. We discuss how environmental cues and monocyte-intrinsic properties may affect their differentiation toward specific functional and phenotypic subsets, the causes of monocyte expansion and reduction in cancer, their metabolic requirements, and the potential effect on tumor immunity.

Complement downregulation promotes an inflammatory signature that renders colorectal cancer susceptible to immunotherapy.

BACKGROUND AND AIMS: The role of inflammatory immune responses in colorectal cancer (CRC) development and response to therapy is a matter of intense debate. While inflammation is a known driver of CRC, inflammatory immune infiltrates are a positive prognostic factor in CRC and predispose to response to immune checkpoint blockade (ICB) therapy. Unfortunately, over 85% of CRC cases are primarily unresponsive to ICB due to the absence of an immune infiltrate, and even the cases that show an initial immune infiltration can become refractory to ICB. The identification of therapy supportive immune responses in the field has been partially hindered by the sparsity of suitable mouse models to recapitulate the human disease. In this study, we aimed to understand how the dysregulation of the complement anaphylatoxin C3a receptor (C3aR), observed in subsets of patients with CRC, affects the immune responses, the development of CRC, and response to ICB therapy. METHODS: We use a comprehensive approach encompassing analysis of publicly available human CRC datasets, inflammation-driven and newly generated spontaneous mouse models of CRC, and multiplatform high-dimensional analysis of immune responses using microbiota sequencing, RNA sequencing, and mass cytometry. RESULTS: We found that patients' regulation of the complement C3aR is associated with epigenetic modifications. Specifically, downregulation of C3ar1 in human CRC promotes a tumor microenvironment characterized by the accumulation of innate and adaptive immune cells that support antitumor immunity. In addition, in vivo studies in our newly generated mouse model revealed that the lack of C3a in the colon activates a microbiota-mediated proinflammatory program which promotes the development of tumors with an immune signature that renders them responsive to the ICB therapy. CONCLUSIONS: Our findings reveal that C3aR may act as a previously unrecognized checkpoint to enhance antitumor immunity in CRC. C3aR can thus be exploited to overcome ICB resistance in a larger group of patients with CRC.

Chronic complement dysregulation drives neuroinflammation after traumatic brain injury: a transcriptomic study.

Activation of the complement system propagates neuroinflammation and brain damage early and chronically after traumatic brain injury (TBI). The complement system is complex and comprises more than 50 components, many of which remain to be characterized in the normal and injured brain. Moreover, complement therapeutic studies have focused on a limited number of histopathological outcomes, which while informative, do not assess the effect of complement inhibition on neuroprotection and inflammation in a comprehensive manner. Using high throughput gene expression technology (NanoString), we simultaneously analyzed complement gene expression profiles with other neuroinflammatory pathway genes at different time points after TBI. We additionally assessed the effects of complement inhibition on neuropathological processes. Analyses of neuroinflammatory genes were performed at days 3, 7, and 28 post injury in male C57BL/6 mice following a controlled cortical impact injury. We also characterized the expression of 59 complement genes at similar time points, and also at 1- and 2-years post injury. Overall, TBI upregulated the expression of markers of astrogliosis, immune cell activation, and cellular stress, and downregulated the expression of neuronal and synaptic markers from day 3 through 28 post injury. Moreover, TBI upregulated gene expression across most complement activation and effector pathways, with an early emphasis on classical pathway genes and with continued upregulation of C2, C3 and C4 expression 2 years post injury. Treatment using the targeted complement inhibitor, CR2-Crry, significantly ameliorated TBI-induced transcriptomic changes at all time points. Nevertheless, some immune and synaptic genes remained dysregulated with CR2-Crry treatment, suggesting adjuvant anti-inflammatory and neurotropic therapy may confer additional neuroprotection. In addition to characterizing complement gene expression in the normal and aging brain, our results demonstrate broad and chronic dysregulation of the complement system after TBI, and strengthen the view that the complement system is an attractive target for TBI therapy.

Preinvasive Colorectal Lesions of African Americans Display an Immunosuppressive Signature Compared to Caucasian Americans.

BACKGROUND: African Americans (AAs) have higher colorectal cancer (CRC) incidence and mortality rate than Caucasian Americans (CAs). Recent studies suggest that immune responses within CRCs contribute to the disparities. If racially distinct immune signatures are present in the early phases of carcinogenesis, they could be used to develop interventions to prevent or slow disease. METHODS: We selected a convenience sample of 95 patients (48 CAs, 47 AAs) with preinvasive colorectal adenomas from the surgical pathology laboratory at the Medical University of South Carolina. Using immunofluorescent-conjugated antibodies on tissue slides from the lesions, we quantified specific immune cell populations: mast cells (CD117+), Th17 cells (CD4+RORC+), and NK cell ligand (MICA/B) and inflammatory cytokines, including IL-6, IL-17A, and IFN-γ. We compared the mean density counts (MDCs) and density rate ratios (RR) and 95% CI of immune markers between AAs to CAs using negative binomial regression analysis. We adjusted our models for age, sex, clinicopathologic characteristics (histology, location, dysplasia), and batch. RESULTS: We observed no racial differences in age or sex at the baseline endoscopic exam. AAs compared to CAs had a higher prevalence of proximal adenomas (66% vs. 40%) and a lower prevalence of rectal adenomas (11% vs. 23%) (p =0.04) but no other differences in pathologic characteristics. In age, sex, and batch adjusted models, AAs vs. CAs had lower RRs for cells labeled with IFNγ (RR 0.50 (95% CI 0.32-0.81); p=0.004) and NK cell ligand (RR 0.67 (0.43-1.04); p=0.07). In models adjusted for age, sex, and clinicopathologic variables, AAs had reduced RRs relative to CAs for CD4 (p=0.02), NK cell ligands (p=0.01), Th17 (p=0.005), mast cells (p=0.04) and IFN-γ (p< 0.0001). CONCLUSIONS: Overall, the lower RRs in AAs vs. CAs suggests reduced effector response capacity and an immunosuppressive ('cold') tumor environment. Our results also highlight the importance of colonic location of adenoma in influencing these differences; the reduced immune responses in AAs relative to CAs may indicate impaired immune surveillance in early carcinogenesis. Future studies are needed to understand the role of risk factors (such as obesity) in influencing differences in immune responses by race.

High Throughput Multi-Omics Approaches for Clinical Trial Evaluation and Drug Discovery.

High throughput single cell multi-omics platforms, such as mass cytometry (cytometry by time-of-flight; CyTOF), high dimensional imaging (>6 marker; Hyperion, MIBIscope, CODEX, MACSima) and the recently evolved genomic cytometry (Citeseq or REAPseq) have enabled unprecedented insights into many biological and clinical questions, such as hematopoiesis, transplantation, cancer, and autoimmunity. In synergy with constantly adapting new single-cell analysis approaches and subsequent accumulating big data collections from these platforms, whole atlases of cell types and cellular and sub-cellular interaction networks are created. These atlases build an ideal scientific discovery environment for reference and data mining approaches, which often times reveals new cellular disease networks. In this review we will discuss how combinations and fusions of different -omic workflows on a single cell level can be used to examine cellular phenotypes, immune effector functions, and even dynamic changes, such as metabolomic state of different cells in a sample or even in a defined tissue location. We will touch on how pre-print platforms help in optimization and reproducibility of workflows, as well as community outreach. We will also shortly discuss how leveraging single cell multi-omic approaches can be used to accelerate cellular biomarker discovery during clinical trials to predict response to therapy, follow responsive cell types, and define novel druggable target pathways. Single cell proteome approaches already have changed how we explore cellular mechanism in disease and during therapy. Current challenges in the field are how we share these disruptive technologies to the scientific communities while still including new approaches, such as genomic cytometry and single cell metabolomics.

Gut vascular barrier impairment leads to intestinal bacteria dissemination and colorectal cancer metastasis to liver.

Metastasis is facilitated by the formation of a "premetastatic niche," which is fostered by primary tumor-derived factors. Colorectal cancer (CRC) metastasizes mainly to the liver. We show that the premetastatic niche in the liver is induced by bacteria dissemination from primary CRC. We report that tumor-resident bacteria Escherichia coli disrupt the gut vascular barrier (GVB), an anatomical structure controlling bacterial dissemination along the gut-liver axis, depending on the virulence regulator VirF. Upon GVB impairment, bacteria disseminate to the liver, boost the formation of a premetastatic niche, and favor the recruitment of metastatic cells. In training and validation cohorts of CRC patients, we find that the increased levels of PV-1, a marker of impaired GVB, is associated with liver bacteria dissemination and metachronous distant metastases. Thus, PV-1 is a prognostic marker for CRC distant recurrence and vascular impairment, leading to liver metastases.

Microbiome as Mediator of Diet on Colorectal Cancer Risk: The Role of Vitamin D, Markers of Inflammation and Adipokines.

Obesity and diet are associated with colorectal cancer (CRC) risk, and microbiome could mediate this risk factor. To investigate this interaction, we performed a case-control study (34 CRC cases and 32 controls) and analyzed fecal microbiota composition using 16S rRNA metabarcoding and sub-sequential shotgun analyses of genomic bacterial DNA to evaluate the role of microbiome and diet in CRC etiology, taking into account vitamin D and other risk biomarkers. Dietary habits were evaluated using a short questionnaire. Multivariate methods for data integration and mediation analysis models were used to investigate causal relationships. CRC cases were significantly more often deficient in vitamin D than controls (p = 0.04); FokI and CYP24A1 polymorphism frequency were different between cases and controls (p = 0.03 and p = 0.02, respectively). A diet poor in fatty fish and rich in carbohydrates was found to be significantly associated with CRC risk (p = 0.011). The mediation analysis confirmed the significant role of the microbiome in mediating CRC risk-increasing levels of Bifidobacteria/Escherichia genera ratio, an indicator of "healthy" intestinal microbiome, can overcome the effect of diet on CRC risk (p = 0.03). This study suggests that microbiome mediates the diet effect on CRC risk, and that vitamin D, markers of inflammation, and adipokines are other factors to consider in order to achieve a better knowledge of the whole carcinogenic process.

Circulating neutrophil subsets in advanced lung cancer patients exhibit unique immune signature and relate to prognosis.

The accumulation of circulating low-density neutrophils (LDN) has been described in cancer patients and associated with tumor-supportive properties, as opposed to the high-density neutrophils (HDN). Here we aimed to evaluate the clinical significance of circulating LDN in lung cancer patients, and further assessed its diagnostic vs prognostic value. Using mass cytometry (CyTOF), we identified major subpopulations within the circulating LDN/HDN subsets and determined phenotypic modulations of these subsets along tumor progression. LDN were highly enriched in the low-density (LD) fraction of advanced lung cancer patients (median 7.0%; range 0.2%-80%, n = 64), but not in early stage patients (0.7%; 0.05%-6%; n = 35), healthy individuals (0.8%; 0%-3.5%; n = 15), or stable chronic obstructive pulmonary disease (COPD) patients (1.2%; 0.3%-7.4%, n = 13). Elevated LDN (>10%) remarkably related with poorer prognosis in late stage patients. We identified three main neutrophil subsets which proportions are markedly modified in cancer patients, with CD66b+ /CD10low /CXCR4+ /PDL1inter subset almost exclusively found in advanced lung cancer patients. We found substantial variability in subsets between patients, and demonstrated that HDN and LDN retain a degree of inherent spontaneous plasticity. Deep phenotypic characterization of cancer-related circulating neutrophils and their modulation along tumor progression is an important advancement in understanding the role of myeloid cells in lung cancer.

Thrombin contributes to cancer immune evasion via proteolysis of platelet-bound GARP to activate LTGF-ß.

Cancer-associated thrombocytosis and high concentrations of circulating transforming growth factor-ß1 (TGF-ß1) are frequently observed in patients with progressive cancers. Using genetic and pharmacological approaches, we show a direct link between thrombin catalytic activity and release of mature TGF-ß1 from platelets. We found that thrombin cleaves glycoprotein A repetitions predominant (GARP), a cell surface docking receptor for latent TGF-ß1 (LTGF-ß1) on platelets, resulting in liberation of active TGF-ß1 from the GARP-LTGF-ß1 complex. Furthermore, systemic inhibition of thrombin obliterates TGF-ß1 maturation in platelet releasate and rewires the tumor microenvironment toward favorable antitumor immunity, which translates into efficient cancer control either alone or in combination with programmed cell death 1-based immune checkpoint blockade therapy. Last, we demonstrate that soluble GARP and GARP-LTGF-ß1 complex are present in the circulation of patients with cancer. Together, our data reveal a mechanism of cancer immune evasion that involves thrombin-mediated GARP cleavage and the subsequent TGF-ß1 release from platelets. We propose that blockade of GARP cleavage is a valuable therapeutic strategy to overcome cancer's resistance to immunotherapy.

Endogenous murine microbiota member Faecalibaculum rodentium and its human homologue protect from intestinal tumour growth.

The microbiota has been shown to promote intestinal tumourigenesis, but a possible anti-tumourigenic effect has also been postulated. Here, we demonstrate that changes in the microbiota and mucus composition are concomitant with tumourigenesis. We identified two anti-tumourigenic strains of the microbiota-Faecalibaculum rodentium and its human homologue, Holdemanella biformis-that are strongly under-represented during tumourigenesis. Reconstitution of ApcMin/+ or azoxymethane- and dextran sulfate sodium-treated mice with an isolate of F. rodentium (F. PB1) or its metabolic products reduced tumour growth. Both F. PB1 and H. biformis produced short-chain fatty acids that contributed to control protein acetylation and tumour cell proliferation by inhibiting calcineurin and NFATc3 activation in mouse and human settings. We have thus identified endogenous anti-tumourigenic bacterial strains with strong diagnostic, therapeutic and translational potential.

GARP Dampens Cancer Immunity by Sustaining Function and Accumulation of Regulatory T Cells in the Colon.

Activated regulatory T (Treg) cells express the surface receptor glycoprotein-A repetitions predominant (GARP), which binds and activates latent TGFß. How GARP modulates Treg function in inflammation and cancer remains unclear. Here we demonstrate that loss of GARP in Treg cells leads to spontaneous inflammation with highly activated CD4+ and CD8+ T cells and development of enteritis. Treg cells lacking GARP were unable to suppress pathogenic T-cell responses in multiple models of inflammation, including T-cell transfer colitis. GARP-/- Treg cells were significantly reduced in the gut and exhibited a reduction in CD103 expression, a colon-specific migratory marker. In the colitis-associated colon cancer model, GARP on Treg cells dampened immune surveillance, and mice with GARP-/- Treg cells exhibited improved antitumor immunity. Thus, GARP empowers the functionality of Treg cells and their tissue-specific accumulation, highlighting the importance of cell surface TGFß in Treg function and GARP as a potential therapeutic target for colorectal cancer therapy.Significance: These findings uncover functions of membrane-bound TGFß and GARP that tune the activity of Treg cells, highlighting a potential treatment strategy in autoimmune diseases and cancer.

Therapeutic faecal microbiota transplantation controls intestinal inflammation through IL10 secretion by immune cells.

Alteration of the gut microbiota has been associated with different gastrointestinal disorders. Normobiosis restoration by faecal microbiota transplantation (FMT) is considered a promising therapeutic approach, even if the mechanisms underlying its efficacy are at present largely unknown. Here we sought to elucidate the functional effects of therapeutic FMT administration during experimental colitis on innate and adaptive immune responses in the intestinal mucosa. We show that therapeutic FMT reduces colonic inflammation and initiates the restoration of intestinal homeostasis through the simultaneous activation of different immune-mediated pathways, ultimately leading to IL-10 production by innate and adaptive immune cells, including CD4+ T cells, iNKT cells and Antigen Presenting Cells (APC), and reduces the ability of dendritic cells, monocytes and macrophages to present MHCII-dependent bacterial antigens to colonic T cells. These results demonstrate the capability of FMT to therapeutically control intestinal experimental colitis and poses FMT as a valuable therapeutic option in immune-related pathologies.

Gone with the Antibody.

Bacterial metabolites can reach distant organs, and in this issue of Immunity, Uchimura et al. (2018) show a fast systemic distribution of microbiota metabolites. This distribution is controlled by antibodies that accelerate bacterial transit through the small intestine, resulting in reduced local and systemic metabolite penetration and attenuation of immune responses.

Author Correction: High-dimensional single-cell analysis predicts response to anti-PD-1 immunotherapy.

In the version of this article initially published, Figs. 5a,c and 6a were incorrect because of an error in a metadata spreadsheet that led to the healthy donor patient 2 (HD2) samples being used twice in the analysis of baseline samples and in the analysis at 12 weeks of anti-PD-1 therapy, while HD3 samples had not been used.

High-dimensional single-cell analysis predicts response to anti-PD-1 immunotherapy.

Immune-checkpoint blockade has revolutionized cancer therapy. In particular, inhibition of programmed cell death protein 1 (PD-1) has been found to be effective for the treatment of metastatic melanoma and other cancers. Despite a dramatic increase in progression-free survival, a large proportion of patients do not show durable responses. Therefore, predictive biomarkers of a clinical response are urgently needed. Here we used high-dimensional single-cell mass cytometry and a bioinformatics pipeline for the in-depth characterization of the immune cell subsets in the peripheral blood of patients with stage IV melanoma before and after 12 weeks of anti-PD-1 immunotherapy. During therapy, we observed a clear response to immunotherapy in the T cell compartment. However, before commencing therapy, a strong predictor of progression-free and overall survival in response to anti-PD-1 immunotherapy was the frequency of CD14+CD16-HLA-DRhi monocytes. We confirmed this by conventional flow cytometry in an independent, blinded validation cohort, and we propose that the frequency of monocytes in PBMCs may serve in clinical decision support.

CyTOF workflow: differential discovery in high-throughput high-dimensional cytometry datasets.

High-dimensional mass and flow cytometry (HDCyto) experiments have become a method of choice for high-throughput interrogation and characterization of cell populations. Here, we present an updated R-based pipeline for differential analyses of HDCyto data, largely based on Bioconductor packages. We computationally define cell populations using FlowSOM clustering, and facilitate an optional but reproducible strategy for manual merging of algorithm-generated clusters. Our workflow offers different analysis paths, including association of cell type abundance with a phenotype or changes in signalling markers within specific subpopulations, or differential analyses of aggregated signals. Importantly, the differential analyses we show are based on regression frameworks where the HDCyto data is the response; thus, we are able to model arbitrary experimental designs, such as those with batch effects, paired designs and so on. In particular, we apply generalized linear mixed models or linear mixed models to analyses of cell population abundance or cell-population-specific analyses of signaling markers, allowing overdispersion in cell count or aggregated signals across samples to be appropriately modeled. To support the formal statistical analyses, we encourage exploratory data analysis at every step, including quality control (e.g., multi-dimensional scaling plots), reporting of clustering results (dimensionality reduction, heatmaps with dendrograms) and differential analyses (e.g., plots of aggregated signals).