Scott N. Furlan

Comprehensive single-cell transcriptional profiling of a multicellular organism

Sequencing each cell of the nematode Single-cell sequencing is challenging owing to the limited biological material available in an individual cell and the high cost of sequencing across multiple cells. Cao et al. developed a two-step combinatorial barcoding method to profile both single-cell and single-nucleus transcriptomes without requiring physical isolation of each cell. The authors profiled almost 50,000 single cells from an individual Caenorhabditis elegans larval stage and were able to identify and recover information from different, even rare, cell types. Science, this issue p. 661 Single-cell combinatorial indexing RNA sequencing achieves more than 50-fold cellular coverage of a developing nematode worm. To resolve cellular heterogeneity, we developed a combinatorial indexing strategy to profile the transcriptomes of single cells or nuclei, termed sci-RNA-seq (single-cell combinatorial indexing RNA sequencing). We applied sci-RNA-seq to profile nearly 50,000 cells from the nematode Caenorhabditis elegans at the L2 larval stage, which provided >50-fold “shotgun” cellular coverage of its somatic cell composition. From these data, we defined consensus expression profiles for 27 cell types and recovered rare neuronal cell types corresponding to as few as one or two cells in the L2 worm. We integrated these profiles with whole-animal chromatin immunoprecipitation sequencing data to deconvolve the cell type–specific effects of transcription factors. The data generated by sci-RNA-seq constitute a powerful resource for nematode biology and foreshadow similar atlases for other organisms.

Programmed death ligand-1 expression on donor T cells drives graft-versus-host disease lethality

Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1-/- donors. PD-L1-deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1-/- donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell-mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD.

Transcriptome analysis of GVHD reveals aurora kinase A as a targetable pathway for disease prevention

Transcriptomic profiling of primate T cells during acute graft-versus-host disease reveals signaling pathways that when inhibited, ameliorate disease. Dawn of new graft-versus-host disease therapies Hematopoietic stem cell transplant (HCT) is a common therapy for patients with damaged bone marrow or immunodeficiencies. However, HCT has its own risks: In cases where the donor is not a perfect match to the recipient, immune cells derived from the graft can attack their new home. Furlan et al. examined the gene expression profile of nonhuman primate T cells during acute graft-versus-host disease (GVHD). The transcriptomics signatures specific for alloreactive T cells identified pathways altered during acute GVHD that could serve as therapeutic targets. The authors then examined one target in particular, aurora kinase A, and demonstrated that pharmacologic inhibition could improve survival in a mouse model of GVHD. Graft-versus-host disease (GVHD) is the most common complication of hematopoietic stem cell transplant (HCT). However, our understanding of the molecular pathways that cause this disease remains incomplete, leading to inadequate treatment strategies. To address this, we measured the gene expression profile of nonhuman primate (NHP) T cells during acute GVHD. Utilizing microarray technology, we measured the expression profiles of CD3+ T cells from five cohorts: allogeneic transplant recipients receiving (i) no immunoprophylaxis (No Rx), (ii) sirolimus monotherapy (Siro), (iii) tacrolimus-methotrexate (Tac-Mtx), as well as (iv) autologous transplant recipients (Auto) and (v) healthy controls (HC). This comparison allowed us to identify transcriptomic signatures specific for alloreactive T cells and determine the impact of both mTOR (mechanistic target of rapamycin) and calcineurin inhibition on GVHD. We found that the transcriptional profile of unprophylaxed GVHD was characterized by significant perturbation of pathways regulating T cell proliferation, effector function, and cytokine synthesis. Within these pathways, we discovered potentially druggable targets not previously implicated in GVHD, prominently including aurora kinase A (AURKA). Utilizing a murine GVHD model, we demonstrated that pharmacologic inhibition of AURKA could improve survival. Moreover, we found enrichment of AURKA transcripts both in allo-proliferating T cells and in sorted T cells from patients with clinical GVHD. These data provide a comprehensive elucidation of the T cell transcriptome in primate acute GVHD and suggest that AURKA should be considered a target for preventing GVHD, which, given the many available AURKA inhibitors in clinical development, could be quickly deployed for the prevention of GVHD.

Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing

Conventional methods for profiling the molecular content of biological samples fail to resolve heterogeneity that is present at the level of single cells. In the past few years, single cell RNA sequencing has emerged as a powerful strategy for overcoming this challenge. However, its adoption has been limited by a paucity of methods that are at once simple to implement and cost effective to scale massively. Here, we describe a combinatorial indexing strategy to profile the transcriptomes of large numbers of single cells or single nuclei without requiring the physical isolation of each cell (Single cell Combinatorial Indexing RNA-seq or sci-RNA-seq). We show that sci-RNA-seq can be used to efficiently profile the transcriptomes of tens-of-thousands of single cells per experiment, and demonstrate that we can stratify cell types from these data. Key advantages of sci-RNA-seq over contemporary alternatives such as droplet-based single cell RNA-seq include sublinear cost scaling, a reliance on widely available reagents and equipment, the ability to concurrently process many samples within a single workflow, compatibility with methanol fixation of cells, cell capture based on DNA content rather than cell size, and the flexibility to profile either cells or nuclei. As a demonstration of sci-RNA-seq, we profile the transcriptomes of 42,035 single cells from C. elegans at the L2 stage, effectively 50-fold “shotgun cellular coverage” of the somatic cell composition of this organism at this stage. We identify 27 distinct cell types, including rare cell types such as the two distal tip cells of the developing gonad, estimate consensus expression profiles and define cell-type specific and selective genes. Given that C. elegans is the only organism with a fully mapped cellular lineage, these data represent a rich resource for future methods aimed at defining cell types and states. They will advance our understanding of developmental biology, and constitute a major step towards a comprehensive, single-cell molecular atlas of a whole animal.

CD8-predominant T-cell CNS infiltration accompanies GVHD in primates and is improved with immunoprophylaxis.

To the editor: A recently published article in Blood showed that, in mice, the central nervous system (CNS) is a target of graft-versus-host-disease (GVHD).[1][1] However, diagnosing CNS GVHD in the clinic is challenging,[2][2][⇓][3]-[4][4] and until now, evidence in a large-animal model has been

The Knife's Edge of Tolerance: Inducing Stable Multilineage Mixed Chimerism But With A Significant Risk of CMV Reactivation and Disease in Rhesus Macaques

Although stable mixed‐hematopoietic chimerism induces robust immune tolerance to solid organ allografts in mice, the translation of this strategy to large animal models and to patients has been challenging. We have previously shown that in MHC‐matched nonhuman primates (NHPs), a busulfan plus combined belatacept and anti‐CD154‐based regimen could induce long‐lived myeloid chimerism, but without T cell chimerism. In that setting, donor chimerism was eventually rejected, and tolerance to skin allografts was not achieved. Here, we describe an adaptation of this strategy, with the addition of low‐dose total body irradiation to our conditioning regimen. This strategy has successfully induced multilineage hematopoietic chimerism in MHC‐matched transplants that was stable for as long as 24 months posttransplant, the entire length of analysis. High‐level T cell chimerism was achieved and associated with significant donor‐specific prolongation of skin graft acceptance. However, we also observed significant infectious toxicities, prominently including cytomegalovirus (CMV) reactivation and end‐organ disease in the setting of functional defects in anti‐CMV T cell immunity. These results underscore the significant benefits that multilineage chimerism‐induction approaches may represent to transplant patients as well as the inherent risks, and they emphasize the precision with which a clinically successful regimen will need to be formulated and then validated in NHP models.

Combined OX40L and mTOR blockade controls effector T cell activation while preserving Treg reconstitution after transplant

OX40L blockade synergizes with sirolimus to prevent GVHD by controlling effector T cell activation while supporting Treg reconstitution. Tackling T cells in GVHD Graft-versus-host disease (GVHD) after stem cell transplantation is mediated by effector T cells derived from donor stem cells, but GVHD can also be abrogated by donor-derived regulatory T cells. GVHD prophylaxis ideally should then allow regulatory T cell responses while inhibiting effector T cells. Tkachev et al. now provide very promising results in a nonhuman primate model, which suggest that such therapy is possible. They used mTOR inhibition in combination with OX40L blockade, which resulted in reduced damaging T cell reconstitution but preserved regulatory T cell activity. The combination therapy also led to a considerable survival benefit. These findings support testing of this therapy in patients. A critical question facing the field of transplantation is how to control effector T cell (Teff) activation while preserving regulatory T cell (Treg) function. Standard calcineurin inhibitor–based strategies can partially control Teffs, but breakthrough activation still occurs, and these agents are antagonistic to Treg function. Conversely, mechanistic target of rapamycin (mTOR) inhibition with sirolimus is more Treg-compatible but is inadequate to fully control Teff activation. In contrast, blockade of OX40L signaling has the capacity to partially control Teff activation despite maintaining Treg function. We used the nonhuman primate graft-versus-host disease (GVHD) model to probe the efficacy of combinatorial immunomodulation with sirolimus and the OX40L-blocking antibody KY1005. Our results demonstrate significant biologic activity of KY1005 alone (prolonging median GVHD-free survival from 8 to 19.5 days), as well as marked, synergistic control of GVHD with KY1005 + sirolimus (median survival time, >100 days; P < 0.01 compared to all other regimens), which was associated with potent control of both TH/TC1 (T helper cell 1/cytotoxic T cell 1) and TH/TC17 activation. Combined administration also maintained Treg reconstitution [resulting in an enhanced Treg/Teff ratio (40% over baseline) in the KY1005/sirolimus cohort compared to a 2.9-fold decrease in the unprophylaxed GVHD cohort]. This unique immunologic signature resulted in transplant recipients that were able to control GVHD for the length of analysis and to down-regulate donor/recipient alloreactivity despite maintaining anti–third-party responses. These data indicate that combined OX40L blockade and sirolimus represents a promising strategy to induce immune balance after transplant and is an important candidate regimen for clinical translation.

Chimeric Antigen Receptor T Cell–Mediated Neurotoxicity in Nonhuman Primates

Chimeric antigen receptor (CAR) T-cell immunotherapy has revolutionized the treatment of refractory leukemias and lymphomas, but is associated with significant toxicities, namely cytokine release syndrome (CRS) and neurotoxicity. A major barrier to developing therapeutics to prevent CAR T cell-mediated neurotoxicity is the lack of clinically relevant models. Accordingly, we developed a rhesus macaque (RM) model of neurotoxicity via adoptive transfer of autologous CD20-specific CAR T cells. Following cyclophosphamide lymphodepletion, CD20 CAR T cells expand to 272 to 4,450 cells/μL after 7 to 8 days and elicit CRS and neurotoxicity. Toxicities are associated with elevated serum IL6, IL8, IL1RA, MIG, and I-TAC levels, and disproportionately high cerebrospinal fluid (CSF) IL6, IL2, GM-CSF, and VEGF levels. During neurotoxicity, both CD20 CAR and non-CAR T cells accumulate in the CSF and in the brain parenchyma. This RM model demonstrates that CAR T cell-mediated neurotoxicity is associated with proinflammatory CSF cytokines and a pan-T cell encephalitis.Significance: We provide the first immunologically relevant, nonhuman primate model of B cell-directed CAR T-cell therapy-mediated CRS and neurotoxicity. We demonstrate CAR and non-CAR T-cell infiltration in the CSF and in the brain during neurotoxicity resulting in pan-encephalitis, accompanied by increased levels of proinflammatory cytokines in the CSF. Cancer Discov; 8(6); 750-63. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 663.

CD28 blockade controls T cell activation to prevent graft-versus-host disease in primates

Controlling graft-versus-host disease (GVHD) remains a major unmet need in stem cell transplantation, and new, targeted therapies are being actively developed. CD28-CD80/86 costimulation blockade represents a promising strategy, but targeting CD80/CD86 with CTLA4-Ig may be associated with undesired blockade of coinhibitory pathways. In contrast, targeted blockade of CD28 exclusively inhibits T cell costimulation and may more potently prevent GVHD. Here, we investigated FR104, an antagonistic CD28-specific pegylated-Fab′, in the nonhuman primate (NHP) GVHD model and completed a multiparameter interrogation comparing it with CTLA4-Ig, with and without sirolimus, including clinical, histopathologic, flow cytometric, and transcriptomic analyses. We document that FR104 monoprophylaxis and combined prophylaxis with FR104/sirolimus led to enhanced control of effector T cell proliferation and activation compared with the use of CTLA4-Ig or CTLA4-Ig/sirolimus. Importantly, FR104/sirolimus did not lead to a beneficial impact on Treg reconstitution or homeostasis, consistent with control of conventional T cell activation and IL-2 production needed to support Tregs. While FR104/sirolimus had a salutary effect on GVHD-free survival, overall survival was not improved, due to death in the absence of GVHD in several FR104/sirolimus recipients in the setting of sepsis and a paralyzed INF-&ggr; response. These results therefore suggest that effectively deploying CD28 in the clinic will require close scrutiny of both the benefits and risks of extensively abrogating conventional T cell activation after transplant.

Characterization of the immune microenvironment of diffuse intrinsic pontine glioma: implications for development of immunotherapy

Background Diffuse intrinsic pontine glioma (DIPG) is a uniformly fatal CNS tumor diagnosed in 300 American children per year. Radiation is the only effective treatment and extends overall survival to a median of 11 months. Due to its location in the brainstem, DIPG cannot be surgically resected. Immunotherapy has the ability to target tumor cells specifically; however, little is known about the tumor microenvironment in DIPGs. We sought to characterize infiltrating immune cells and immunosuppressive factor expression in pediatric low- and high-grade gliomas and DIPG. Methods Tumor microarrays were stained for infiltrating immune cells. RNA was isolated from snap-frozen tumor tissue and Nanostring analysis performed. DIPG and glioblastoma cells were co-cultured with healthy donor macrophages, T cells, or natural killer (NK) cells, and flow cytometry and cytotoxicity assays performed to characterize the phenotype and function, respectively, of the immune cells. Results DIPG tumors do not have increased macrophage or T-cell infiltration relative to nontumor control, nor do they overexpress immunosuppressive factors such as programmed death ligand 1 and/or transforming growth factor β1. H3.3-K27M DIPG cells do not repolarize macrophages, but are not effectively targeted by activated allogeneic T cells. NK cells lysed all DIPG cultures. Conclusions DIPG tumors have neither a highly immunosuppressive nor inflammatory microenvironment. Therefore, major considerations for the development of immunotherapy will be the recruitment, activation, and retention of tumor-specific effector immune cells.