Search

Movie M2. Representative example of a CAR8 cell from the S2 subgroup.

Table ST1: Glossary of terms used in the paper Figure S1. Genetic modification and expansion of CAR+ T cells. Figure S2. In vitro expansion of CAR+ T cells. Figure S3. High-throughput cytotoxicity assay for monitoring T-cell target cell interactions in nanowell grids. Figure S4. CD19 expression on NALM-6 tumor cells or CD19+EL4 target cells as determined by immunofluoresecent staining. Figure S5. Composite micrographs illustrating representative examples of the interactions between single CAR+ T cells (E) and one or more NALM-6 tumor (T) cells. Figure S6. Increased probability of killing by individual CAR+ T cells at higher target cell densities. Figure S7. Donut plots summarizing the outcomes of the interaction between individual CAR8 cells and 1-3 CD19+-NALM-6 tumor cells. Figure S8. Timelapse Imaging Microscopy In Nanowell Grids (TIMING). Figure S9. At an E:T ratio of 1:1, identification of subgroups of killer CAR8 cells based on their motility and contact behavior with tumor cells. Figure S10. At an E:T of 1:1, the total duration of conjugation prior to NALM-6 tumor cell killing is no different for the CAR8 cells in the different subgroups. Figure S11. At an E:T ratio of 1:1, CAR8 cells in the S1 subgroup, demonstrate: (A) drop in motility, and (B) increased circularization upon conjugation to NALM-6 tumor cell. Figure S12. At an E:T ratio of 1:1, CAR8 cells in the different subgroups demonstrate different frequencies and kinetics of AICD subsequent to the interactions with NALM-6 cells. Figure S13. At an E:T ratio of 1:2-5, multi-killer CAR8 cells demonstrate no significant differences in their duration of conjugation prior to killing multiple NALM-6 tumor cells. Figure S14. Despite the increased crowding because of higher tumor cell density, multi-killer CAR8 cells displayed greater motility when conjugated to tumor cell in comparison to single-killer CAR8 cells that encountered only a single tumor cell. Figure S15. At an E:T ratio of 1:1, identification of subgroups of killer CAR4 cells based on their motility and contact behavior with tumor cells. Figure S16. At an E:T of 1:1, the total duration of conjugation prior to NALM-6 tumor cell killing is significantly longer for CAR4 cells in S2 subgroup in comparison to subgroups S1 and S3. Figure S17. At an E:T of 1:1, CAR4 cells in S2 subgroup induce apoptosis in tumor cells with delayed kinetics in comparison to CAR8 cells in the S2 subgroup. Figure S18. At an E:T ratio of 1:2-5, multi-killer CAR4 cells demonstrate increased circularization upon contact with one or more NALM-6 tumor cells. Figure S19. The ability of individual CAR4 cells to simultaneously conjugate to multiple NALM-6 tumor cells increases as the number of tumor cells within the nanowell increases. Figure S20. Comparison of the killing efficiency of individual single killer CAR+ T cells (E:T 1:1) with multi-killer CAR+ T cells (E:T 1:2-5) that killed multiple NALM-6 tumor cells.

T cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) for the investigational treatment of B-cell malignancies comprise a heterogeneous population, and their ability to persist and participate in serial killing of tumor cells is a predictor of therapeutic success. We implemented Timelapse Imaging Microscopy in Nanowell Grids (TIMING) to provide direct evidence that CD4+CAR+ T cells (CAR4 cells) can engage in multikilling via simultaneous conjugation to multiple tumor cells. Comparisons of the CAR4 cells and CD8+CAR+ T cells (CAR8 cells) demonstrate that, although CAR4 cells can participate in killing and multikilling, they do so at slower rates, likely due to the lower granzyme B content. Significantly, in both sets of T cells, a minor subpopulation of individual T cells identified by their high motility demonstrated efficient killing of single tumor cells. A comparison of the multikiller and single-killer CAR+ T cells revealed that the propensity and kinetics of T-cell apoptosis were modulated by the number of functional conjugations. T cells underwent rapid apoptosis, and at higher frequencies, when conjugated to single tumor cells in isolation, and this effect was more pronounced on CAR8 cells. Our results suggest that the ability of CAR+ T cells to participate in multikilling should be evaluated in the context of their ability to resist activation-induced cell death. We anticipate that TIMING may be used to rapidly determine the potency of T-cell populations and may facilitate the design and manufacture of next-generation CAR+ T cells with improved efficacy. Cancer Immunol Res; 3(5); 473–82. ©2015 AACR.See related commentary by June, p. 470

Movie M4. Representative example of a CAR8 cell that kills multiple tumor cells.

Movie M1. Representative example of a CAR8 cell from the S1 subgroup.

Movie M3. Representative example of a CAR8 cell from the S3 subgroup.

Movie M5. Representative example of a CAR4 cell that kills multiple tumor cells.

Adoptive immunotherapy with T cells expressing chimeric antigen receptors (CARs) for B-cell malignancies serves as a model for identifying subsets with superior clinical activity. We profiled the infusion products (IP) of 16 patients with large B-cell lymphoma (LBCL) using an integrated suite of single-cell assays to reveal the therapeutic potential of CD19-specific CAR+ T cells. Timelapse imaging microscopy in nanowell grids (TIMING) profiling revealed that T cells from responders showed migration (persistent motion for at least one body length), and migration was associated with serial killing capacity. In addition, confocal microscopy revealed that migration is linearly correlated with both mitochondrial volume and lysosomal volume; and scRNA-seq demonstrated that T cells from responders were enriched in pathways related to T-cell killing, migration and actin cytoskeleton, and TCR clustering. A marker-free sorting strategy enriched T cells with migratory capacity and validated serial killing, bioenergetics, and in vivo efficacy. In aggregate, we demonstrate that migration is a cell-intrinsic biomarker independent of CAR design or biomanufacturing, desired in the bioactivity of CAR+ T cells associated with clinical antitumor efficacy.

The in vivo persistence of adoptively transferred T cells is predictive of anti-tumor response. Identifying functional properties of infused T cells that lead to in vivo persistence and tumor eradication has remained elusive. We profiled CD19-specific CAR T cells that comprise the infusion products used to treat large B cell lymphomas using high-throughput single-cell technologies based on Timelapse Imaging Microscopy In Nanowell Grids (TIMING) that integrates killing, cytokine secretion, and transcriptional profiling. Our results show that the directional migration of CD19-specific CAR T cells is correlated with polyfunctionality. We identified that CD2 on T cells is associated with directional migration and that the interaction between CD2 on T cells and CD58 on lymphoma cells accelerates killing and serial killing. Consistent with this, we observed elevated CD58 expression on pre-treatment tumor samples in patients with relapsed or refractory large B cell lymphomas treated with CD19-specific CAR T cell therapy was associated with complete clinical response and survival. These results highlight the importance of studying dynamic T-cell tumor cell interactions in identifying optimal antitumor responses.KEY POINTS- Profiling patient infusion products revealed that polyfunctional CAR T cells show directional migration, which is associated with higher CD2 expression- The ligand for CD2, CD58 is expressed at higher levels in the tumors of lymphoma patients who respond better to CAR T cell treatment

BackgroundAdoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive.MethodsTo identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling.ResultsWith the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107apos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice.ConclusionsBroadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit.

T-cell therapy with specificity redirected through chimeric antigen receptors (CARs) has shown efficacy for the treatment of hematologic malignancies. Although treatment with CAR T cell can result in high response rates, the properties of the cells that comprise the cellular infusion product, associated with clinical benefit are incompletely understood. We utilized a suite of high-throughput single-cell assays including single-cell RNA-sequencing (scRNA-seq); confocal microscopy; and Timelapse Imaging Microscopy In Nanowell Grids (TIMING). TIMING profiling of a cohort of 16 patients showed that persistent motility of T cells in the presence of tumor cells was associated with both serial killing capacity and polyfunctionality. Confocal microscopy on these same T cells revealed that persistent motility is linearly correlated with both mitochondrial volume and lysosomal number. ScRNA-seq demonstrated that T cells from responders were enriched in pathways related to T-cell proliferative capacity; interferon responses; and a distinct cluster of pathways related to actin cytoskeleton and migration. We employed a marker-free sorting strategy for enriching T cells with persistent motility. RNA-seq on sorted motile T cells showed an enrichment of the core motility signature. These motile T cells also demonstrated superior in vivo anti-leukemia efficacy in comparison to unsorted T cells. Lastly, we also confirmed the association with increased persistent motility and killing of CAR T cells across diverse CARs. In aggregate, our data identified that independent of CAR design or biomanufacturing, persistent motility serves as a selectable cell-intrinsic biomarker, desired in the bioactivity of expanded CAR+ T cells.

Genetically engineered T cells that express chimeric antigen receptors (CAR+) are heterogeneous and thus, understanding the immunotherapeutic efficacy remains a challenge in adoptive cell therapy. We developed a high-throughput single-cell methodology, Timelapse Imaging Microscopy In Nanowell Grids (TIMING) to monitor interactions between immune cells and tumor cells in vitro. Using TIMING we demonstrated that CD4+ CAR+ T cells participate in multi-killing and benefit from improved resistance to activation induced cell death in comparison to CD8+ CAR+ T cells. For both subsets of cells, effector cell fate at the single-cell level was dependent on functional activation through multiple tumor cells.

Objective To obtain the comprehensive transcriptome profile of human citrulline-specific B cells from patients with rheumatoid arthritis (RA). Methods Citrulline- and hemagglutinin-specific B cells were sorted by flow cytometry using peptide-streptavidin conjugates from the peripheral blood of RA patients and healthy individuals. The transcriptome profile of the sorted cells was obtained by RNA-sequencing, and expression of key protein molecules was evaluated by aptamer-based SOMAscan assay and flow cytometry. The ability of these proteins to effect differentiation of osteoclasts and proliferation and migration of synoviocytes was examined by in vitro functional assays. Results Citrulline-specific B cells, in comparison to citrulline-negative B cells, from patients with RA differentially expressed the interleukin-15 receptor α (IL-15Rα) gene as well as genes related to protein citrullination and cyclic AMP signaling. In analyses of an independent cohort of cyclic citrullinated peptide-seropositive RA patients, the expression of IL-15Rα protein was enriched in citrulline-specific B cells from the patients' peripheral blood, and surprisingly, all B cells from RA patients were capable of producing the epidermal growth factor ligand amphiregulin (AREG). Production of AREG directly led to increased migration and proliferation of fibroblast-like synoviocytes, and, in combination with anti-citrullinated protein antibodies, led to the increased differentiation of osteoclasts. Conclusion To the best of our knowledge, this is the first study to document the whole transcriptome profile of autoreactive B cells in any autoimmune disease. These data identify several genes and pathways that may be targeted by repurposing several US Food and Drug Administration-approved drugs, and could serve as the foundation for the comparative assessment of B cell profiles in other autoimmune diseases.

High-throughput flow cytometry is an attractive platform for the analysis of adoptive cellular therapies such as chimeric antigen receptor T cell therapy (CAR-T) because it allows for the concurrent measurement of T cell-dependent cellular cytotoxicity (TDCC) and the functional characterization of engineered T cells with respect to percentage of CAR transduction, T cell phenotype, and measurement of T cell function such as activation in a single assay. The use of adherent tumor cell lines can be challenging in these flow-based assays. Here, we present the development of a high-throughput flow-based assay to measure TDCC for a CAR-T construct co-cultured with multiple adherent tumor cell lines. We describe optimal assay conditions (such as adherent cell dissociation techniques to minimize impact on cell viability) that result in robust cytotoxicity assays. In addition, we report on the concurrent use of T cell transduction and activation antibody panels (CD25) that provide further dissection of engineered T cell function. In conclusion, we present the development of a high-throughput flow cytometry method allowing for in vitro interrogation of solid tumor, targeting CAR-T cell-mediated cytotoxicity, CAR transduction, and engineered T cell characterization in a single assay.

Motivation: There is a need for effective automated methods for profiling dynamic cell–cell interactions with single-cell resolution from high-throughput time-lapse imaging data, especially, the interactions between immune effector cells and tumor cells in adoptive immunotherapy. Results: Fluorescently labeled human T cells, natural killer cells (NK), and various target cells (NALM6, K562, EL4) were co-incubated on polydimethylsiloxane arrays of sub-nanoliter wells (nanowells), and imaged using multi-channel time-lapse microscopy. The proposed cell segmentation and tracking algorithms account for cell variability and exploit the nanowell confinement property to increase the yield of correctly analyzed nanowells from 45% (existing algorithms) to 98% for wells containing one effector and a single target, enabling automated quantification of cell locations, morphologies, movements, interactions, and deaths without the need for manual proofreading. Automated analysis of recordings from 12 different experiments demonstrated automated nanowell delineation accuracy >99%, automated cell segmentation accuracy >95%, and automated cell tracking accuracy of 90%, with default parameters, despite variations in illumination, staining, imaging noise, cell morphology, and cell clustering. An example analysis revealed that NK cells efficiently discriminate between live and dead targets by altering the duration of conjugation. The data also demonstrated that cytotoxic cells display higher motility than non-killers, both before and during contact. Contact: broysam@central.uh.edu or nvaradar@central.uh.edu Supplementary information: Supplementary data are available at Bioinformatics online.

International audience; Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.

Although the contribution of B-cell derived autoreactive antibodies to rheumatoid arthritis (RA) has been studied extensively, the autoantibody-independent roles of B cells in the progression of the disease is not well-defined. Here we present the first comprehensive transcriptome profile of human autoreactive B cells in an autoimmune disease by performing RNA-sequencing of citrulline-specific B cells from RA patients. In order to facilitate a comprehensive understanding of the profile of these citrulline-specific (RA-CCPPOS) B cells, we performed comparative analyses to both citrulline-negative (RA-CCPNEG) B cells from the same donors, and identified 431 differentially expressed genes (DEGs); and hemagglutinin-specific (HA) B cells from healthy individuals and identified 1658 DEGs. Three-way comparisons of these B cell populations demonstrated that RA-CCPPOS B cells, in comparison to the RA-CCPNEG B cells, demonstrate a potential role in protein citrullination and inflammation; RA-CCPPOS B cells in comparison to HA-specific B cells demonstrate RA-specific signatures like the expression of pro-inflammatory cytokines, chemokines, costimulatory molecules and B-cell activation cascades; and all B cells from RA patients demonstrated a significant impact of the multitude of TNF signaling pathways. Furthermore, transcription factor profiling suggested that cyclic AMP (cAMP) related pathways and downstream signaling molecules are selectively enriched in RA-CCPPOS cells in comparison to the other two B cell subsets. We advanced the understanding of the citrulline reactive B cells in RA pathophysiology by documenting and validating two novel observations in independent cohorts of patients: (1) the expression of IL15Rα is restricted to citrulline-specific cells within RA patients and the concentration of soluble IL15Rα is elevated in the sera of RA patients, (2) B cells from RA patients are capable of producing epidermal growth factor ligand, amphiregulin (AREG) which in turn has a direct impact on the mechanistic effectors of RA, osteoclasts and fibroblastlike synoviocytes (FLS). Overall, our comprehensive dataset identifies several existing FDA-approved drugs that can potentially be repurposed for RA and can serve as a foundation for studying the multi-faceted roles of B cells in other autoimmune diseases.

Dendritic cells are major APCs that can efficiently prime immune responses. However, the roles of skin-resident Langerhans cells (LCs) in eliciting immune responses have not been fully understood. In this study, we demonstrate for the first time, to our knowledge, that LCs in cynomolgus macaque skin are capable of inducing antiviral-specific immune responses in vivo. Targeting HIV-Gag or influenza hemagglutinin Ags to skin LCs using recombinant fusion proteins of anti-Langerin Ab and Ags resulted in the induction of the viral Ag-specific responses. We further demonstrated that such Ag-specific immune responses elicited by skin LCs were greatly enhanced by TLR ligands, polyriboinosinic polyribocytidylic acid, and R848. These enhancements were not due to the direct actions of TLR ligands on LCs, but mainly dependent on TNF-α secreted from macrophages and neutrophils recruited to local tissues. Skin LC activation and migration out of the epidermis are associated with macrophage and neutrophil infiltration into the tissues. More importantly, blocking TNF-α abrogated the activation and migration of skin LCs. This study highlights that the cross-talk between innate immune cells in local tissues is an important component for the establishment of adaptive immunity. Understanding the importance of local immune networks will help us to design new and effective vaccines against microbial pathogens.

T cells engineered to express chimeric antigen receptor (CAR) targeting CD19 have shown promising clinical responses in patients with certain hematologic malignancies, however, it is desirable to be able to enrich cells with enhanced anti-tumor efficacy prior to infusion. We utilized a suite of high-throughput technologies with single-cell resolution, including Timelapse Imaging Microscopy In Nanowell Grids (TIMING) that integrates cytokine profiling to reveal that persistent motility of CD19- specific CAR T cells is correlated to desirable polyfunctionality (elimination of tumor cells and cytokine secretion), contributing to anti-tumor effects. We implemented a marker-free Boyden chamber-based method to enrich CAR+ T cells with persistent motility (motile cell). Integration of transcriptomic profiling, immune phenotyping and metabolism demonstrated that motile cells are more naïve-like with higher oxidative metabolism and spare respiratory capacity. Our result also revealed that the master metabolic regulator AMP kinase (AMPK) is required for CAR+ T cells with high motility. We used a xenograft leukemia mouse model (CD19+ NALM-6) and validated that the motile cells have enhanced persistence and superior anti-cancer effect in vivo compared to the parental un-sorted population. Collectively, our multi-dimensional results demonstrated that persistent motility is a selectable biomarker of expanded CAR+ T cell bioactivity.

The isolation of human monoclonal antibodies (hmAb) has emerged as a versatile platform in a wide variety of contexts ranging from vaccinology to therapeutics. In particular, the presence of high titers of circulating auto-antibodies is implicated in the pathology and outcome of autoimmune diseases. Therefore, the molecular characterization of these hmAb provides an avenue to understanding the pathogenesis of autoimmune diseases. Additionally, the phenotype of the auto-reactive B cells may have direct relevance for therapeutic intervention. In this report, we describe a high-throughput single-cell assay, microengraving, for the screening, characterization and isolation of anti-citrullinated protein antibodies (ACPA) from peripheral blood mononuclear cells (PBMC) of rheumatoid arthritis (RA) patients. Stimulated B cells are profiled at the single-cell level in a large array of sub-nanoliter nanowells (∼10(5)), assessing both the phenotype of the cells and their ability to secrete cyclic-citrullinated peptide (CCP)-specific antibodies. Single B cells secreting ACPA are retrieved by automated micromanipulation, and amplification of the immunoglobulin (Ig) heavy and light chains is performed prior to recombinant expression. The methodology offers a simple, rapid and low-cost platform for isolation of auto-reactive antibodies from low numbers of input cells and can be easily adapted for isolation and characterization of auto-reactive antibodies in other autoimmune diseases.

Natural killer (NK) cells are a highly heterogeneous population of innate lymphocytes that constitute our first line of defense against several types of tumors and microbial infections. Understanding the heterogeneity of these lymphocytes requires the ability to integrate their underlying phenotype with dynamic functional behaviors. We have developed and validated a single-cell methodology that integrates cellular phenotyping and dynamic cytokine secretion based on nanowell arrays and bead-based molecular biosensors. We demonstrate the robust passivation of the polydimethylsiloxane (PDMS)-based nanowells arrays with polyethylene glycol (PEG) and validated our assay by comparison to enzyme-linked immunospot (ELISPOT) assays. We used numerical simulations to optimize the molecular density of antibodies on the surface of the beads as a function of the capture efficiency of cytokines within an open-well system. Analysis of hundreds of individual human peripheral blood NK cells profiled ex vivo revealed that CD56dimCD16+ NK cells are immediate secretors of interferon gamma (IFN-γ) upon activation by phorbol 12-myristate 13-acetate (PMA) and ionomycin (< 3 h), and that there was no evidence of cooperation between NK cells leading to either synergistic activation or faster IFN-γ secretion. Furthermore, we observed that both the amount and rate of IFN-γ secretion from individual NK cells were donor-dependent. Collectively, these results establish our methodology as an investigational tool for combining phenotyping and real-time protein secretion of individual cells in a high-throughput manner.

The pivotal role of DCs in initiating immune responses led to their use as vaccine vectors. However, the relationship between DC subsets involved in antigen presentation and the type of elicited immune responses underlined the need for the characterization of the DCs generated in vitro. The phenotypes of tissue-derived APCs from a cynomolgus macaque model for human vaccine development were compared with ex vivo-derived DCs. Monocyte/macrophages predominated in bone marrow (BM) and blood. Myeloid DCs (mDCs) were present in all tested tissues and were more highly represented than plasmacytoid DCs (pDCs). As in human skin, Langerhans cells (LCs) resided exclusively in the macaque epidermis, expressing CD11c, high levels of CD1a and langerin (CD207). Most DC subsets were endowed with tissue-specific combinations of PRRs. DCs generated from CD34+ BM cells (CD34-DCs) were heterogeneous in phenotype. CD34-DCs shared properties (differentiation and PRR) of dermal and epidermal DCs. After injection into macaques, CD34-DCs expressing HIV-Gag induced Gag-specific CD4+ and CD8+ T cells producing IFN-γ, TNF-α, MIP-1β, or IL-2. In high responding animals, the numbers of polyfunctional CD8+ T cells increased with the number of booster injections. This DC-based vaccine strategy elicited immune responses relevant to the DC subsets generated in vitro.

Genetically engineered T cells that express chimeric antigen receptors (CAR(+)) are heterogeneous and thus, understanding the immunotherapeutic efficacy remains a challenge in adoptive cell therapy. We developed a high-throughput single-cell methodology, Timelapse Imaging Microscopy In Nanowell Grids (TIMING) to monitor interactions between immune cells and tumor cells in vitro. Using TIMING we demonstrated that CD4(+) CAR(+) T cells participate in multi-killing and benefit from improved resistance to activation induced cell death in comparison to CD8(+) CAR+ T cells. For both subsets of cells, effector cell fate at the single-cell level was dependent on functional activation through multiple tumor cells.

T cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) for the investigational treatment of B-cell malignancies comprise a heterogeneous population, and their ability to persist and participate in serial killing of tumor cells is a predictor of therapeutic success. We implemented Timelapse Imaging Microscopy in Nanowell Grids (TIMING) to provide direct evidence that CD4+CAR+ T cells (CAR4 cells) can engage in multikilling via simultaneous conjugation to multiple tumor cells. Comparisons of the CAR4 cells and CD8+CAR+ T cells (CAR8 cells) demonstrate that, although CAR4 cells can participate in killing and multikilling, they do so at slower rates, likely due to the lower granzyme B content. Significantly, in both sets of T cells, a minor subpopulation of individual T cells identified by their high motility demonstrated efficient killing of single tumor cells. A comparison of the multikiller and single-killer CAR+ T cells revealed that the propensity and kinetics of T-cell apoptosis were modulated by the number of functional conjugations. T cells underwent rapid apoptosis, and at higher frequencies, when conjugated to single tumor cells in isolation, and this effect was more pronounced on CAR8 cells. Our results suggest that the ability of CAR+ T cells to participate in multikilling should be evaluated in the context of their ability to resist activation-induced cell death. We anticipate that TIMING may be used to rapidly determine the potency of T-cell populations and may facilitate the design and manufacture of next-generation CAR+ T cells with improved efficacy. Cancer Immunol Res; 3(5); 473–82. ©2015 AACR. See related commentary by June, p. 470

Non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system has been successfully tested in humans to express a chimeric antigen receptor (CAR) to redirect T-cell specificity to CD19. This system has been modified to (i) improve the design of the CD19-specific CAR and (ii) reduce the time in culture to 14 days. Our previous clinical trials infused T cells expressing a 2nd generation CAR (designated CD19RCD28) with an IgG4-Fc stalk that activated via chimeric CD28 and CD3ζ. To evaluate the length of extracellular domain on function, we tested four CD19-specific CARs with two long [IgG4-Fc (CD19RCD28) and EQ (L235E and N297Q) mutant IgG4-Fc (CD19R*CD28)], medium (CD8α hinge, CD19RCD8CD28) and short (12aa IgG1 hinge, CD19R12aaCD28) stalks which all signaled through chimeric CD28 and CD3ζ endodomains. Generation of our T cells is based on electro-transfer of CARs coded by the SB system and antigen-specific stimulation through activating and K562-derived propagating cells (AaPC) in the presence of exogenous cytokines. After electro-transfer of SB-derived DNA plasmids, T cells were selectively propagated with either a new two-weekly (2x) or standard four-weekly (4x) additions of AaPC. All genetically modified T cells were capable of specific lysis of CD19+ tumor targets and producing IFN-γ in response to CD19+ stimulator cells. Serial killing was tested using massively parallel microscopy to observe single T cells and we observed that CDl9RCD8CD28+ T cells exhibited superior ability to partake in multiple killing events. CAR+ T cells were further tested in vivo for their ability to control CD19+ leukemia in a mouse model of minimal residual disease as well as established disease (Figure A and BFigure A and B). We found that T cells expressing modified CARs (CD19R*CD28, CD19RCD8CD28, CD19R12aaCD28) with reduced ability to bind to Fc gamma receptors (FcγR) were able to control leukemia more efficiently in mice compared to T cells expressing CD19RCD28. The CD19RCD8CD28 CAR was superior in controlling disease in the model of minimal residual disease compared with the CAR design evaluated in our prior clinical trials. T cells expressing CD19R*CD28 and CD19RCD8CD28 were then evaluated in 2x stimulation cycle. Both the 4x CAR+ T cells had similar CAR expression (>70%) whereas the 2x CAR+ T cells exhibited reduced CAR expression (~40%). The 2x CAR+ T cells expressed markers associated with less differentiated state of naive-like and memory T cells when compared to 4x CAR+ T cells, which was supported by measurement of mRNA species using bar-coded probes. The efficacy of the CAR+ T cells was tested in mice bearing established CD19+ leukemia and we observed superior survival in mice receiving the 2x CAR+ T cells compared with the 4x CAR+ T cells (Figure CFigure C). These data depict that length of extracellular domain and its associated binding to FcγR improves T-cell effector functions and that decreasing the time in culture can improve control of leukemia in vivo. These data support the use of cDl9RCD8CD28 testing in a next-generation clinical trial (IND# 16474).View Large Image | Download PowerPoint Slide

Nat. Immunol.; doi:10.1038/ni.3770; corrected online 27 June 2017 In the version of this article initially published online, the labels identifying each plot in Figure 1b were missing. The labels are as follows (left to right): CHO, FcγRI, FcγRIIaR131, FcγRIIaH131, FcγRIIb, FcγRIIIaF158 and FcγRIIIaV158.

The efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effector-mediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cell-mediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia.

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials(1-4). There are several advantages to using CAR(+) T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR(+) T cells in the event of host toxicity(5). Delineating the optimal functions of CAR(+) T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo(6,7). While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets(8). Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes(9). In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

Adoptive cell therapy (ACT) based on the transfer of chimeric antigen receptor (CAR) T cells rendered specific for CD19 has demonstrated significant anti-tumor effects in patients with refractory CD19+ B-cell malignancies. There are however no biomarkers that can predict the potency of T-cell infusion products and thus the ability to enrich functionally superior cells is lacking. Existing clinical data supports at least two important functions of the T cells in mediating tumor regression: (a) immediate cytotoxicity to enable tumor cell killing, and (b) long-term persistence to ensure lasting and durable responses. We demonstrate here that migratory capacity of T cells can be used as a biomarker to segregate functionally superior cells that display enhanced polyfunctionality in vitro and superior tumor control in vivo. The simplicity of sorting cells based on motility and the scalability of the approach imply that these can be routinely adapted in standard GMP settings to enrich for functional T cells among any heterogeneous population. Populations of CD19-specific chimeric antigen receptor (CAR) T cells were biomanufactured using our standard clinical protocol. Timelapse Imaging In Nanowell Grids (TIMING) was used to enable the simultaneous quantification of the interaction between thousands of individual tumor-specific CD8+ T cells and multiple tumor cells. These results demonstrated that IFN-gamma secretion was the most common function elicited upon tumor cell conjugation. We found that CD8+ T cells with killing ability, especially serial killing ability, required shorter durations of target cell conjugation in comparison to IFN-gamma secreting mono-functional cells, indicating rapid synapse termination by T cells capable of killing versus cytokine secretion. Tracking the velocities of these cells by time-lapse imaging revealed that these serial killer T cells (with or without IFN-gamma secretion) had a high out-of-contact basal motility. In order to gain a better understanding of the phenotypic characteristics of the migratory T cells, we performed molecular profiling of T cells identified only by their basal motility (in the absence of tumor cells) using single-cell multiplexed transcriptional profiling, microscopy and flow cytometry, and found that the costimulatory molecules CD2 and CD244, and the chemokine receptor CXCR3 were expressed at higher levels in highly motile T cells compared to the non-motile T cells. In order to test the hypothesis that migratory capacity can serve as a biomarker T cell polyfunctionality, we next sorted motile cells using a modified migration assay, and confirmed that the migrating cells were enriched in highly motile cells. We characterized these cells phenotypically by flow-cytometry and demonstrated that T-cell differentiation markers including CCR7, CD95 and CD45RA were not differentially expressed between the motile and non-motile cells. Consistently however, the migrated cells displayed superior killing and serial killing in comparison to the non-migrated T cells. This superior in vitro polyfunctionality of migratory T cells was confirmed in a mouse xenograft model of leukemia in which motile CAR+ T cells were superior in controlling the disease in comparison to either their non-motile counterparts or the parent unsorted population. In aggregate, these results demonstrate the utility of our TIMING single cell methodology in uncovering not only the dynamic profile of T-cell behavior but also the ability to identify subpopulations of T-cell with enhanced polyfunctionality. Our studies support the use of motility as a surrogate and selective marker of higher CAR+ T cell bioactivity. These results also open up avenues to molecularly engineer T cells for an increased motility that could translate to better in vivo outcomes. Note: This abstract was not presented at the conference. Citation Format: Gabrielle Romain, Harjeet Singh, Ivan Liadi, Jay R Adolacion, Laurence J.N. Cooper, Navin Varadarajan. Migratory T cells demonstrate superior persistence and enhanced tumor control [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B082.

Interferon-γ (IFNγ), a cytokine secreted by effector lymphocytes upon activation, is known to cause upregulation of MHC class I in tumor cells, which has been associated with natural killer (NK) cell inhibition. NK cells are very heterogeneous with respect to receptor expression and their function depends on an interplay between these inhibitory and activating signals. In order to optimize NK cell therapy, it is essential to identify the NK cells expressing the optimal combination of receptors for a given target. In this study we evaluated tumor cell lines from the Pediatric Preclinical Testing Program to determine the impact of IFNγ on their expression of NK cell activating and inhibitory ligands, death receptors, and adhesion molecules using mass cytometry, and their correlation with tumor lysis. We found that IFNγ treatment can significantly increase ICAM-1 and/or HLA-ABC levels together or independently. When HLA-ABC increases without concomitant ICAM-1 upregulation, this is associated with decreased lysis. Conversely, when ICAM-1 upregulation exceeds HLA-ABC upregulation this is associated with increased conjugate formation and better NK cell cytotoxic activity. This increase in cytotoxicity and conjugate formation is lost after blocking ICAM-1. Time-lapse imaging of the NK cell interaction with tumor cells shows that NK cells require less time to form stable contacts with IFNγ treated tumor cells when compared to untreated tumor cells. This suggests that IFNγ induced upregulation of ICAM-1 enhances NK cell cytotoxic activity by reducing the time required for NK cells to form stable contacts with their target.

Strategies to improve vaccine efficacy are still required, especially in the case of chronic infections, including human immunodeficiency virus (HIV). DNA vaccines have potential advantages over conventional vaccines; however, low immunological efficacy has been demonstrated in many experiments involving large animals and in clinical trials. To improve the immunogenicity of DNA vaccines, we have designed a plasmid vector exploiting the binding capacity of the bovine papillomavirus E2 protein and we have used electroporation (EP) to increase DNA uptake after intradermal inoculation. We demonstrated, in nonhuman primates (NHPs), efficient induction of anti-HIV immunity with an improved DNA vaccine vector encoding an artificial fusion protein, consisting of several proteins and selected epitopes from HIV-1. We show that a DNA vaccine delivery method combining intradermal injection and noninvasive EP dramatically increased expression of the vaccine antigen selectively in the epidermis, and our observations strongly suggest the involvement of Langerhans cells in the strength and quality of the anti-HIV immune response. Although the humoral responses to the vaccine were transient, the cellular responses were exceptionally robust and persisted, at high levels, more than 2 years after the last vaccine boost. The immune responses were characterized by the induction of significant proportions of T cells producing both interferon-gamma and interleukin-2 cytokines, in both subpopulations, CD4(+) and CD8(+). This strategy is an attractive approach for vaccination in humans because of its high efficacy and the possible use of newly developed devices for EP.

Background Antiretroviral therapies against HIV infection reduce the plasma viral load but can not eradicate the virus. Dendritic cells (DC) transfected with messenger ribonucleic acids (mRNA) encoding endogenous viral proteins are expected to enhance the HIV specific immune response and are considered as potent therapeutic vaccines. In this context we studied the feasibility and efficiency of mRNA loaded CD34+ derived DC as therapeutic vaccine in SIV infected macaques as a model of HIV infection and AIDS.

Meeting abstracts T cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) for the investigational treatment of B cell malignancies comprise a heterogeneous population, and their ability to persist and participate in serial killing of tumor cells is a predictor of

Adoptive cell therapy (ACT) utilizing chimeric antigen receptor (CAR) T cells rendered specific for CD19 have demonstrated significant anti-tumor effects in patients with CD19+ chronic lymphocytic leukemia (CLL). In spite of the clinical promise of ACT in achieving complete responses, their efficacy remains unpredictable and new approaches are needed to address a priori define the therapeutic potential of T-cell based therapies. In our current work, we characterize the in vitro functionality of CD19-specific (CD19RCD28) CAR+ T cells propagated using artificial antigen presenting cells expressing membrane bound IL-21, by employing a novel methodology single-cell nanowell screening that determines their cytotoxic ability and cytokine secretion capability at single-cell resolution. We show that CAR+ T cells exert specific cytotoxicity against NALM6 cells (31 ± 8 %) when co-incubated at a 1:1 ratio in nanowell containers. Furthermore, single CAR+ T cells were capable of engaging and killing multiple targets; 17 ± 8% of T cells killed two target cells and 9 ± 3% killed three target cells within the 6 hour window of observation. In parallel, microengraving was used to determine the cytokine secretion profile of these same cells. Hierarchical clustering of the two functions indicated that interferon-gamma (IFNγ) secretion is not correlated to cytotoxicity or the ability of T cells to kill multiple target cells. Simultaneously, monitoring apoptosis on CAR+ T cells allowed us to quantify their activation-induced cell death (AICD). CAR+ T cells that secreted IFNγ upon target ligation did not undergo AICD whereas T cells that engaged in repeated killing showed an increased propensity to undergo AICD (p = 0.04). Dynamic time-lapse imaging of the interactions between CAR+ T cells and tumor cells indicated that the majority of CAR+ T cells have high basal motility, form long-lived interactions with tumor cells (50 - 100 min) that lead to motility arrest and subsequent tumor-cell apoptosis. However, contact lifetimes or overall contact duration were not reliable predictors of subsequent tumor-cell apoptosis. Finally, kinetics of serial killing suggest that motile CAR+ T cells that form short-lived contacts exhibit rapid killing with very little motility arrest in vitro. In summary, our SNS based methodology allows the deep functional characterization of clinical grade CAR+ T cells and can be used to: (1) determine in vitro functions of CAR+ T cells that correlate with clinical efficacy and (2) inform CAR design to maximize effector functionality while minimizing AICD.

Meeting abstracts Humanized monoclonal antibodies (mAb) targeting tumor antigens have paved the way to complementary strategies in the treatment of cancer by harnessing the immune system towards a more specific response against tumors. Such mAb can mobilize natural killer (NK) cells function by

Adoptive cell therapy (ACT) utilizing chimeric antigen receptor (CAR) T cells rendered specific for CD19 have demonstrated significant anti-tumor effects in patients with CD19+ chronic lymphocytic leukemia (CLL). In spite of the clinical promise of ACT in achieving complete responses, their efficacy remains unpredictable and new approaches are needed to a priori define the therapeutic potential of T-cell based therapies. In our current work, we characterize the in vitro functionality of CD19-specific (CD19RCD28) CAR+ T cells propagated using artificial antigen presenting cells, by employing a novel methodology single-cell nanowell screening (SNS).We show that CAR+ T cells exert specific cytotoxicity against NALM6 cells (31 ± 8 %) when co-incubated at a 1:1 ratio in nanowell containers. Furthermore, single CAR+ T cells were capable of engaging and killing multiple targets; 17 ± 8% of T cells killed two target cells within the 6 hour window of observation. Hierarchical clustering indicated that interferon-gamma (IFNγ) secretion is not correlated to cytotoxicity of T cells. CAR+ T cells that secreted IFNγ upon target ligation did not undergo activation-induced cell death (AICD) whereas T cells that engaged in repeated killing showed an increased propensity to undergo AICD (p = 0.04). In summary, our SNS based methodology allows the deep functional characterization of clinical grade CAR+ T cells and can be used to determine in vitro functions of CAR+ T cells.

Adoptive cell therapy (ACT) utilizing chimeric antigen receptor (CAR) T cells rendered specific for CD19 have demonstrated significant anti-tumor effects in patients with CD19+ chronic lymphocytic leukemia (CLL) that were considered untreatable, including the use of stem-cell transplantation. In spite of the clinical promise of ACT in achieving complete responses, their efficacy remains unpredictable and new approaches are needed to address a priori define the therapeutic potential of T-cell based therapies. In our current work, we characterize the in vitro functionality of CD19-specific (CD19RCD28) CAR+ T cells propagated using artificial antigen presenting cells expressing membrane bound IL-21, by employing a novel methodology single-cell nanowell screening that determines their cytotoxic ability and cytokine secretion capability at single-cell resolution. We show that CAR+ T cells exert specific cytotoxicity against NALM6 cells (31 ± 8 %) when co-incubated at a 1:1 ratio in nanowell containers. Furthermore, single CAR+ T cells were capable of engaging and killing multiple targets; 17 ± 8% of T cells killed two target cells and 9 ± 3% killed three target cells within the 6 hour window of observation. In parallel, microengraving was used to determine the cytokine secretion profile of these same cells. Hierarchical clustering of the two functions indicated that interferon-gamma (IFNγ) secretion is not correlated to cytotoxicity or the ability of T cells to kill multiple target cells. Simultaneously, monitoring apoptosis on CAR+ T cells allowed us to quantify their activation-induced cell death (AICD). CAR+ T cells that secreted IFNγ upon target ligation did not undergo AICD whereas T cells that engaged in repeated killing showed an increased propensity to undergo AICD (p = 0.04). Finally, automated time-lapse microscopy of hundreds of these effector target interactions allowed us to quantify the kinetics of effector mediated lysis and effector cell velocity profiles before and after lysis. These analyses demonstrate that individual CAR+ T cells demonstrate vastly different individual killing profiles, at least in vitro. In summary, our SNS based methodology allows the deep functional characterization of clinical grade CAR+ T cells and can be used to determine in vitro functions of CAR+ T cells that correlate with clinical efficacy. Citation Format: Navin Varadarajan, Ivan Liadi, Gabrielle Romain, Harjeet Singh, Laurence J.N. Cooper. Quantitative single-cell functional characterization of CD19-specific CAR+ T Cells for immunotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4745. doi:10.1158/1538-7445.AM2013-4745

Meeting abstracts T cells genetically modified to enforce expression of a chimeric antigen receptor (CAR) have shown considerable promise in clinical trials even in tumors refractory to all other treatment methods. In particular, the use of CAR T cells rendered specific for CD19 demonstrated