Molecular cytometry for comprehensive immune profiling.

Molecular cytometry refers to a group of high-parameter technologies for single-cell analysis that share the following traits: (1) combined (multimodal) measurement of protein and transcripts, (2) medium throughput (10-100K cells), and (3) the use of oligonucleotide-tagged antibodies to detect protein expression. The platform can measure over 100 proteins and either hundreds of targeted genes or the whole transcriptome, on a cell-by-cell basis. It is currently one of the most powerful technologies available for immune monitoring. Here, we describe the technology platform (which includes CITE-Seq, REAP-Seq, and AbSeq), provide guidance for its optimization, and discuss advantages and limitations. Finally, we provide some vignettes from studies that demonstrate the application and potential insight that can be gained from molecular cytometry studies.

A Phase 1/2 multicenter trial of DKN-01 as monotherapy or in combination with docetaxel for the treatment of metastatic castration-resistant prostate cancer (mCRPC).

BACKGROUND: Dickkopf-related protein 1 (DKK1) is a Wingless-related integrate site (Wnt) signaling modulator that is upregulated in prostate cancers (PCa) with low androgen receptor expression. DKN-01, an IgG4 that neutralizes DKK1, delays PCa growth in pre-clinical DKK1-expressing models. These data provided the rationale for a clinical trial testing DKN-01 in patients with metastatic castration-resistant PCa (mCRPC). METHODS: This was an investigator-initiated parallel-arm phase 1/2 clinical trial testing DKN-01 alone (monotherapy) or in combination with docetaxel 75 mg/m2 (combination) for men with mCRPC who progressed on ≥1 AR signaling inhibitors. DKK1 status was determined by RNA in-situ expression. The primary endpoint of the phase 1 dose escalation cohorts was the determination of the recommended phase 2 dose (RP2D). The primary endpoint of the phase 2 expansion cohorts was objective response rate by iRECIST criteria in patients treated with the combination. RESULTS: 18 pts were enrolled into the study-10 patients in the monotherapy cohorts and 8 patients in the combination cohorts. No DLTs were observed and DKN-01 600 mg was determined as the RP2D. A best overall response of stable disease occurred in two out of seven (29%) evaluable patients in the monotherapy cohort. In the combination cohort, five out of seven (71%) evaluable patients had a partial response (PR). A median rPFS of 5.7 months was observed in the combination cohort. In the combination cohort, the median tumoral DKK1 expression H-score was 0.75 and the rPFS observed was similar between patients with DKK1 H-score ≥1 versus H-score = 0. CONCLUSION: DKN-01 600 mg was well tolerated. DKK1 blockade has modest anti-tumor activity as a monotherapy for mCRPC. Anti-tumor activity was observed in the combination cohorts, but the response duration was limited. DKK1 expression in the majority of mCRPC is low and did not clearly correlate with anti-tumor activity of DKN-01 plus docetaxel.

Corrigendum: Tumor infiltrating T cell states and checkpoint inhibitor expression in hepatic and pancreatic malignancies.

[This corrects the article DOI: 10.3389/fimmu.2023.1067352.].

Interferon alfa-2b in patients with low-grade lymphomatoid granulomatosis and chemotherapy with DA-EPOCH-R in patients with high-grade lymphomatoid granulomatosis: an open-label, single-centre, phase 2 trial.

BACKGROUND: Lymphomatoid granulomatosis is a rare Epstein-Barr virus-associated B-cell lymphoproliferative disorder with a median overall survival of less than 2 years. In this study, we hypothesised that low-grade lymphomatoid granulomatosis is immune-dependent and high-grade lymphomatoid granulomatosis is immune-independent. On the basis of this hypothesis, we investigated the activity and safety of new treatment with immunotherapy in patients with low-grade disease and standard chemotherapy in patients with high-grade disease. METHODS: In this open-label, single-centre, phase 2 trial, we enrolled patients aged 12 years or older with untreated, or relapsed or refractory lymphomatoid granulomatosis at the National Cancer Institute (National Institutes of Health, Bethesda, MD, USA). Patients with low-grade disease received dose-escalated interferon alfa-2b, starting at 7·5 million international units subcutaneously three times per week for up to 1 year past best response, and patients with high-grade disease received six cycles every 3 weeks of intravenous, dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (DA-EPOCH-R). Starting doses were 50 mg/m2 per day as a continuous intravenous infusion from day 1 to day 4 (96 h) for etoposide; 60 mg/m2 twice daily by mouth from day 1 to day 5 for prednisone; 0·4 mg/m2 per day as a continuous intravenous infusion from day 1 to day 4 (96 h) for vincristine; 750 mg/m2 intravenous on day 5 for cyclophosphamide; 10 mg/m2 per day as a continuous intravenous infusion from day 1 to day 4 (96 h) for doxorubicin; and 375 mg/m2 intravenous on day 1 for rituximab. The doses of doxorubicin, etoposide, and cyclophosphamide were adjusted up or down on the basis of neutrophil and platelet nadirs. Patients with residual or progressive disease after initial therapy crossed over to alternative therapy. The primary endpoint was the proportion of patients who had an overall response and the 5-year progression-free survival after initial or cross-over treatment. Analysis of response included all participants who underwent restaging imaging; safety analysis included all patients who received any dose of study drugs. The trial is open for enrolment and is registered at ClinicalTrials.gov, NCT00001379. FINDINGS: 67 patients were enrolled between Jan 10, 1991, and Sept 5, 2019 (42 [63%] were male). 45 patients received initial treatment with interferon alfa-2b (16 of whom crossed over to DA-EPOCH-R) and 18 received initial treatment with DA-EPOCH-R (eight of whom crossed over to interferon alfa-2b); four underwent surveillance only. After initial treatment with interferon alfa-2b, the overall response was 64% (28 of 44 evaluable patients) with 61% (27 of 44) having a complete response, whereas, after cross-over treatment with interferon alfa-2b, the overall response was 63% (five of eight evaluable patients) with 50% (four of eight) having a complete response. After initial treatment with DA-EPOCH-R, the overall response was 76% (13 of 17 evaluable patients) with 47% (eight of 17) having a complete response, whereas, after cross-over treatment with DA-EPOCH-R, the overall response was 67% (ten of 15 evaluable patients) with 47% (seven of 15) having a complete response. 5-year progression-free survival was 48·5% (95% CI 33·2-62·1) after initial treatment with interferon alfa-2b, 50·0% (15·2-77·5) after cross-over treatment with interferon alfa-2b, 25·4% (8·2-47·2) after initial treatment with DA-EPOCH-R, and 62·5% (34·9-81·1) after cross-over treatment with DA-EPOCH-R. The most common grade 3 or worse adverse events in patients treated with interferon alfa-2b included neutropenia (27 [53%] of 51 patients), lymphopenia (24 [47%]), and leukopenia (24 [47%]). The four most common grade 3 or worse adverse events in patients treated with DA-EPOCH-R included neutropenia (29 [88%] of 33 patients), leukopenia (28 [85%]), infection (18 [55%]), and lymphopenia (17 [52%]). Serious adverse events occurred in 13 (25%) of 51 patients receiving treatment with interferon alfa-2b and 21 (64%) of 33 patients receiving DA-EPOCH-R, with five treatment-related deaths: one thromboembolic, one infection, and one haemophagocytic syndrome with interferon alfa-2b, and one infection and one haemophagocytic syndrome with DA-EPOCH-R. INTERPRETATION: Interferon alfa-2b is efficacious for treating low-grade lymphomatoid granulomatosis and hence reducing progression to high-grade disease, whereas patients with high-grade lymphomatoid granulomatosis showed expected responses to chemotherapy. Uncontrolled immune regulation of Epstein-Barr virus is hypothesised to result in the emergence of low-grade disease after chemotherapy, for which treatment with interferon alfa-2b is efficacious. FUNDING: Intramural Research Programs of the National Cancer Institute and National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Tumor infiltrating T cell states and checkpoint inhibitor expression in hepatic and pancreatic malignancies.

Hepato-pancreatico-biliary (HPB) malignancies are difficult-to-treat and continue to to have a high mortality and significant therapeutic resistance to standard therapies. Immune oncology (IO) therapies have demonstrated efficacy in several solid malignancies when combined with chemotherapy, whereas response rates in pancreatic ductal adenocarcinoma (PDA) are poor. While promising in hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), there remains an unmet need to fully leverage IO therapies to treat HPB tumors. We therefore defined T cell subsets in the tumor microenvironment of HPB patients utilizing a novel, multiparameter flow cytometry and bioinformatics analysis. Our findings quantify the T cell phenotypic states in relation to checkpoint receptor expression. We demonstrate the presence of CD103+ tissue resident memory T cells (TRM), CCR7+ central memory T cells, and CD57+ terminally differentiated effector cells across all HPB cancers, while the anti-tumor function was dampened by expression of multiple co-inhibitory checkpoint receptors. Terminally exhausted T cells lacking co-stimulatory receptors were more prevalent in PDA, whereas partially exhausted T cells expressing both co-inhibitory and co-stimulatory receptors were most prevalent in HCC, especially in early stage. HCC patients had significantly higher TRM with a phenotype that could confer restored activation in response to immune checkpoint therapies. Further, we found a lack of robust alteration in T cell activation state or checkpoint expression in response to chemotherapy in PDA patients. These results support that HCC patients might benefit most from combined checkpoint therapies, whereas efforts other than cytotoxic chemotherapy will likely be necessary to increase overall T cell activation in CCA and PDA for future clinical development.

Single cell multiomic analysis of T cell exhaustion in vitro.

T-cell activation is a key step in the amplification of an immune response. Over the course of an immune response, cells may be chronically stimulated, with some proportion becoming exhausted; an enormous number of molecules are involved in this process. There remain a number of questions about the process, namely: (1) what degree of heterogeneity and plasticity do T-cells exhibit during stimulation? (2) how many unique cell states define chronic stimulation? and (3) what markers discriminate activated from exhausted cells? We addressed these questions by performing single-cell multiomic analysis to simultaneously measure expression of 38 proteins and 399 genes in human T cells expanded in vitro. This approach allowed us to study -with unprecedented depth-how T cells change over the course of chronic stimulation. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states and the identification of a donor-specific subset of terminally differentiated T-cells that would have been otherwise overlooked using canonical cell classification schema. As expected, activation downregulated naïve-cell markers and upregulated effector molecules, proliferation regulators, co-inhibitory and co-stimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation, defined by markers temporarily expressed upon 3 days of stimulation (PD-1, CD69, LTA), markers constitutively expressed throughout chronic activation (CD25, GITR, LGALS1), and markers uniquely up-regulated upon 14 days of stimulation (CD39, ENTPD1, TNFDF10); expression of these markers could be associated with the emergence of short-lived cell types. Notably, different ratios of cells expressing activation or exhaustion markers were measured at each time point. These data reveal the high heterogeneity and plasticity of chronically stimulated T cells. Our study demonstrates the power of a single-cell multiomic approach to comprehensively characterize T-cells and to precisely monitor changes in differentiation, activation, and exhaustion signatures during cell stimulation.

Tonic interferon restricts pathogenic IL-17-driven inflammatory disease via balancing the microbiome.

Maintenance of immune homeostasis involves a synergistic relationship between the host and the microbiome. Canonical interferon (IFN) signaling controls responses to acute microbial infection, through engagement of the STAT1 transcription factor. However, the contribution of tonic levels of IFN to immune homeostasis in the absence of acute infection remains largely unexplored. We report that STAT1 KO mice spontaneously developed an inflammatory disease marked by myeloid hyperplasia and splenic accumulation of hematopoietic stem cells. Moreover, these animals developed inflammatory bowel disease. Profiling gut bacteria revealed a profound dysbiosis in the absence of tonic IFN signaling, which triggered expansion of TH17 cells and loss of splenic Treg cells. Reduction of bacterial load by antibiotic treatment averted the TH17 bias and blocking IL17 signaling prevented myeloid expansion and splenic stem cell accumulation. Thus, tonic IFNs regulate gut microbial ecology, which is crucial for maintaining physiologic immune homeostasis and preventing inflammation.

Illuminating the immunopathology of SARS-CoV-2.

Over a remarkably short period of time, a great deal of knowledge about severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection has been acquired, through the focused and cooperative effort of the international scientific community. Much has become known about how the immune response is coordinated to fight infection, and how it becomes dysregulated in severe disease. In this review, we take an in-depth look at the many immune features associated with the host response to SARS-CoV2, as well as those that appear to mark severe disease.

A Cytometrist's Guide to Coordinating and Performing Effective COVID-19 Research.

Cytometry is playing a crucial role in addressing the COVID-19 pandemic. In this commentary-written by a variety of stakeholders in the cytometry, immunology, and infectious disease communities-we review cytometry's role in the COVID-19 response and discuss workflow issues critical to planning and executing effective research in this emerging field. We discuss sample procurement and processing, biosafety, technology options, data sharing, and the translation of research findings into clinical environments. © 2020 International Society for Advancement of Cytometry.

Nivolumab and ipilimumab are associated with distinct immune landscape changes and response-associated immunophenotypes.

BACKGROUNDThe reshaping of the immune landscape by nivolumab (NIVO) and ipilimumab (IPI) and its relation to patient outcomes is not well described.METHODSWe used high-parameter flow cytometry and a computational platform, CytoBrute, to define immunophenotypes of up to 15 markers to assess peripheral blood samples from metastatic melanoma patients receiving sequential NIVO > IPI or IPI > NIVO (Checkmate-064).RESULTSThe 2 treatments were associated with distinct immunophenotypic changes and had differing profiles associated with response. Only 2 immunophenotypes were shared but had opposing relationships to response/survival. To understand the impact of sequential treatment on response/survival, phenotypes that changed after the initial treatment and differentiated response in the other cohort were identified. Immunophenotypic changes occurring after NIVO were predominately associated with response to IPI > NIVO, but changes occurring after IPI were predominately associated with progression after NIVO > IPI. Among these changes, CD4+CD38+CD39+CD127-GARP- T cell subsets were increased after IPI treatment and were negatively associated with response/survival for the NIVO > IPI cohort.CONCLUSIONCollectively, these data suggest that the impact of IPI and NIVO on the immunophenotypic landscape of patients is distinct and that the impact of IPI may be associated with resistance to subsequent NIVO therapy, consistent with poor outcomes in the IPI > NIVO cohort of Checkmate-064.

High-Parameter Single-Cell Analysis.

Thousands of transcripts and proteins confer function and discriminate cell types in the body. Using high-parameter technologies, we can now measure many of these markers at once, and multiple platforms are now capable of analysis on a cell-by-cell basis. Three high-parameter single-cell technologies have particular potential for discovering new biomarkers, revealing disease mechanisms, and increasing our fundamental understanding of cell biology. We review these three platforms (high-parameter flow cytometry, mass cytometry, and a new class of technologies called integrated molecular cytometry platforms) in this article. We describe the underlying hardware and instrumentation, the reagents involved, and the limitations and advantages of each platform. We also highlight the emerging field of high-parameter single-cell data analysis, providing an accessible overview of the data analysis process and choice of tools.

A deadly dance: the choreography of host-pathogen interactions, as revealed by single-cell technologies.

Pathogens have numerous mechanisms by which they replicate within a host, who in turn responds by developing innate and adaptive immune countermeasures to limit disease. The advent of high-content single-cell technologies has facilitated a greater understanding of the properties of host cells harboring infection, the host's pathogen-specific immune responses, and the mechanisms pathogens have evolved to escape host control. Here we review these advances and argue for greater inclusion of higher resolution single-cell technologies into approaches for defining immune evasion mechanisms by pathogens.

Kinetics of adult hematopoietic stem cell differentiation in vivo.

Adult hematopoiesis has been studied in terms of progenitor differentiation potentials, whereas its kinetics in vivo is poorly understood. We combined inducible lineage tracing of endogenous adult hematopoietic stem cells (HSCs) with flow cytometry and single-cell RNA sequencing to characterize early steps of hematopoietic differentiation in the steady-state. Labeled cells, comprising primarily long-term HSCs and some short-term HSCs, produced megakaryocytic lineage progeny within 1 wk in a process that required only two to three cell divisions. Erythroid and myeloid progeny emerged simultaneously by 2 wk and included a progenitor population with expression features of both lineages. Myeloid progenitors at this stage showed diversification into granulocytic, monocytic, and dendritic cell types, and rare intermediate cell states could be detected. In contrast, lymphoid differentiation was virtually absent within the first 3 wk of tracing. These results show that continuous differentiation of HSCs rapidly produces major hematopoietic lineages and cell types and reveal fundamental kinetic differences between megakaryocytic, erythroid, myeloid, and lymphoid differentiation.

What to expect when you're expecting.

Applying systems immunology to study human pregnancy.

Simultaneous epitope and transcriptome measurement in single cells.

High-throughput single-cell RNA sequencing has transformed our understanding of complex cell populations, but it does not provide phenotypic information such as cell-surface protein levels. Here, we describe cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), a method in which oligonucleotide-labeled antibodies are used to integrate cellular protein and transcriptome measurements into an efficient, single-cell readout. CITE-seq is compatible with existing single-cell sequencing approaches and scales readily with throughput increases.

Combined single-cell quantitation of host and SIV genes and proteins ex vivo reveals host-pathogen interactions in individual cells.

CD4 T cells harboring HIV-1/SIV represent a formidable hurdle to eradicating infection, and yet their detailed phenotype remains unknown. Here we integrate two single-cell technologies, flow cytometry and highly multiplexed quantitative RT-PCR, to characterize SIV-infected CD4 T cells directly ex vivo. Within individual cells, we correlate the cellular phenotype, in terms of host protein and RNA expression, with stages of the viral life cycle defined by combinatorial expression of viral RNAs. Spliced RNA+ infected cells display multiple memory and activation phenotypes, indicating virus production by diverse CD4 T cell subsets. In most (but not all) cells, progressive infection accompanies post-transcriptional downregulation of CD4 protein, while surface MHC class I is largely retained. Interferon-stimulated genes were also commonly upregulated. Thus, we demonstrate that combined quantitation of transcriptional and post-transcriptional regulation at the single-cell level informs in vivo mechanisms of viral replication and immune evasion.