Navin Varadarajan

High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire

Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (VH and VL) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 104 capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion VH:VL linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of VH:VL pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets—peripheral blood IgG+ B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccination.

A high-throughput single-cell analysis of human CD8 + T cell functions reveals discordance for cytokine secretion and cytolysis

CD8+ T cells are a key component of the adaptive immune response to viral infection. An inadequate CD8+ T cell response is thought to be partly responsible for the persistent chronic infection that arises following infection with HIV. It is therefore critical to identify ways to define what constitutes an adequate or inadequate response. IFN-γ production has been used as a measure of T cell function, but the relationship between cytokine production and the ability of a cell to lyse virus-infected cells is not clear. Moreover, the ability to assess multiple CD8+ T cell functions with single-cell resolution using freshly isolated blood samples, and subsequently to recover these cells for further functional analyses, has not been achieved. As described here, to address this need, we have developed a high-throughput, automated assay in 125-pl microwells to simultaneously evaluate the ability of thousands of individual CD8+ T cells from HIV-infected patients to mediate lysis and to produce cytokines. This concurrent, direct analysis enabled us to investigate the correlation between immediate cytotoxic activity and short-term cytokine secretion. The majority of in vivo primed, circulating HIV-specific CD8+ T cells were discordant for cytolysis and cytokine secretion, notably IFN-γ, when encountering cognate antigen presented on defined numbers of cells. Our approach should facilitate determination of signatures of functional variance among individual effector CD8+ T cells, including those from mucosal samples and those induced by vaccines.

Rapid, efficient functional characterization and recovery of HIV-specific human CD8+ T cells using microengraving

The nature of certain clinical samples (tissue biopsies, fluids) or the subjects themselves (pediatric subjects, neonates) often constrain the number of cells available to evaluate the breadth of functional T-cell responses to infections or therapeutic interventions. The methods most commonly used to assess this functional diversity ex vivo and to recover specific cells to expand in vitro usually require more than 106 cells. Here we present a process to identify antigen-specific responses efficiently ex vivo from 104–105 single cells from blood or mucosal tissues using dense arrays of subnanoliter wells. The approach combines on-chip imaging cytometry with a technique for capturing secreted proteins—called “microengraving”—to enumerate antigen-specific responses by single T cells in a manner comparable to conventional assays such as ELISpot and intracellular cytokine staining. Unlike those assays, however, the individual cells identified can be recovered readily by micromanipulation for further characterization in vitro. Applying this method to assess HIV-specific T-cell responses demonstrates that it is possible to establish clonal CD8+ T-cell lines that represent the most abundant specificities present in circulation using 100- to 1,000-fold fewer cells than traditional approaches require and without extensive genotypic analysis a priori. This rapid (<24 h), efficient, and inexpensive process should improve the comparative study of human T-cell immunology across ages and anatomic compartments.

Highly active and selective endopeptidases with programmed substrate specificities

A family of engineered endopeptidases has been created that is capable of cleaving a diverse array of peptide sequences with high selectivity and catalytic efficiency (kcat/KM > 10(40 M(- 1) s(- 1)). By screening libraries with a selection-counterselection substrate method, protease variants were programmed to recognize amino acids having altered charge, size and hydrophobicity properties adjacent to the scissile bond of the substrate, including GluArg, a specificity that to our knowledge has not been observed among natural proteases. Members of this artificial protease family resulted from a relatively small number of amino acid substitutions that (at least in one case) proved to be epistatic.

Antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells

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.

Individual Motile CD4+ T Cells Can Participate in Efficient Multikilling through Conjugation to Multiple Tumor Cells

Liadi, Singh, and colleagues used Timelapse Imaging Microscopy In Nanowell Grids (TIMING) to show that CD4+ CD19-chimeric antigen receptor (CAR+) T cells participate in multikilling of tumor cells with slower kinetics of killing than CD8+CAR+ T cells, but high motility subgroups of both T-cell subsets have similar kinetics. 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

An Engineered Protease that Cleaves Specifically after Sulfated Tyrosine

Sulfated tyrosines are present in a wide array of proteins, such as G-protein-coupled receptors (GPCRs),[1] anticoagulation/coagulation factors,[2,3] antibodies,[4] and bioactive peptides, such as phyllokinin, phytosulfokine, and cholecystokinin.[1] The post-translational addition of a sulfate group to tyrosine residues on peptides and proteins is catalyzed by membrane-bound tyrosylprotein sulfotransferases (TPSTs)[5] in the trans-Golgi network.[5] Sulfation occurs following protein translocation to the endoplasmic reticulum, and thus, there is spatial separation between the two common forms of post-translationally modified tyrosine residues, namely, phosphorylation in the cytosol, and sulfation in the extracellular space.[3] Although tyrosine-O-sulfation plays a critical role in protein–protein interactions, in cell function, and in certain disease states,[3] the elucidation of sulfation sites on proteins and peptides, and consequently the understanding of their function, is challenging.[1] There are no consensus sequences for tyrosine sulfation other than the presence of neighboring acidic residues. Furthermore, the sulfate group is hydrolyzed at low-pH conditions typically used for chemical analysis[6] and during analysis under positive/negative mode MS/MS.[7,8] We have been pursuing an enzyme-engineering-based[9,10] approach to detect post-translationally modified tyrosines by altering the substrate specificity of the Escherichia coli outer membrane protease OmpT to cleave only those proteins with modified tyrosine residues. To realize this goal, an engineered protease must cleave at a modified tyrosine residue while being able to discriminate between the chemically similar sulfotyrosine (sTyr) and phosphotyrosine (pTyr) modifications. Herein is reported a highly active engineered OmpT variant (kcat/KM > 1 × 105M−1s−1, where kcat is the catalytic rate constant and KM is the Michaelis constant) exhibiting specific recognition of sulfotyrosine in the P1 position. Significantly, this OmpT variant showed greater than 200-fold and tenfold preferences in favor of sulfotyrosine over phosphotyrosine and unmodified tyrosine, respectively. Recently, a rational engineering effort to expand the substrate selectivity of the bacterial protease subtilisin BPN′ resulted in an enzyme capable of cleaving substrates containing either sulfo- or phosphotyrosine, with the latter being preferred by roughly a factor of two.[11] To engineer OmpT to recognize selectively sulfotyrosine-containing substrates, a sulfotyrosine selection peptide was synthesized, in which the sulfotyrosine residue was flanked by a fluorophore (BODIPY) and a positively-charged tail on one side, and a quencher (QSY 7) on the other.[12] Cleavage at the sulfotyrosine residue by enzyme variants displayed on the surface of E. coli resulted in capture of the positively-charged fluorescent moiety. Simultaneous counterselection using a zwitterionic, fluorescently labeled (tetramethyl rhodamine) peptide containing the wild-type (WT) OmpT-preferred dibasic sequence (ArgArg) was used to eliminate nonspecific protease variants (Figure 1a). Figure 1 a) Two-color flow-cytometric scheme for the isolation of sulfotyrosine-specific OmpT variants. BD =BODIPY, TMR =tetramethylrhodamine, Q =QSY 7. b) Fluorescence profiles (emission at λ =530 nm with a 30 nm filter) of cells expressing no enzyme ... A partial saturation library (the targeted amino acid is randomly encoded for by either the wild-type or NNS codon (N =guanine, adenine, thymine, or cytosine; S =guanine or cytosine) targeting the 21 amino acids (Figure S1 in the Supporting Information) lining the entire OmpT active site[13] (excluding the putative catalytic residues Asp83, Asp85, Asp210, and His212) was constructed by oligonucleotide-based gene assembly in which degenerate NNS oligonucleotides (90 mol%) were mixed with WT oligonucleotides (10 mol%).[12] The library was cloned into pDUCE19, a plasmid that expresses OmpT under the control of its native promoter, and transformed into electrocompetent E. coli MC1061 to generate 3 × 108 transformants. Plasmid was isolated from pooled cells and retransformed into E. coli BL21(DE3), an ompT ompP deficient strain. The cells were grown for 6–8 h at 37°C to an optical density at λ =600 nm OD600 ≈ 2. A 1 mL aliquot of the culture (ca. 109 cells) was washed and resuspended in 1% sucrose with 20 nM selection substrate 1a and 100 nM 5 a for ten minutes and sorted by flow cytometry (MoFlo, Dako, Fort Collins, CO). Gates were set based on forward/side scatter and FL-1 (BODIPY fluorescence)/FL-2 (TMR fluorescence) to collect E. coli cells expressing OmpT variants with high BODIPY and low TMR fluorescence. After five rounds of regrowth and sorting, the isolated cells were plated onto lysogeny broth (Difco) agar plates containing ampicillin. Three unique clones (designated sT1, sT2, and sT3) were isolated after the final round of sorting. Fluorescence analysis of cells labeled with 1a–4a demonstrated that the enzyme variants were selective, but the overall fluorescence with 1a was not indicative of a highly active enzyme variant (Figure 1b). To isolate an OmpT variant exhibiting more efficient hydrolysis of 1a, the three clones above were backcrossed with WT OmpT using DNA shuffling[14] to yield a library of 2 × 106 independent transformants. After three rounds of flow cytometric sorting as above, six clones were isolated, and DNA sequencing revealed three unique variants. One clone (designated sT4) exhibited a selective fluorescence profile when labeled individually with 1a–5a. Clone sT4 contains a total of nine amino acid changes, and notably, both aromatic and basic amino acids had been introduced in the putative S1 binding pocket (Table 1). Table 1 Amino acid changes and kinetic parameters for WT OmpT and the sulfotyrosine variant sT4, measured at room temperature (25°C). The sT4 variant was expressed and purified (to greater than 90% purity) with approximately the same yield as the WT protein (Figure S2 in the Supporting Information) using extraction with n-octylglucoside,[15] and the kinetics of hydrolysis of unlabeled peptide substrates 1 b–6 b were determined (Figure 1c). In accordance with its fluorescence profile, sT4 demonstrated efficient hydrolysis of the sulfotyrosine peptide 1b, with kcat/KM = 1.1 ± 0.2 × 105M−1s−1 (Figure S3 in the Supporting Information). The engineered enzyme did not exhibit Michaelis–Menten behavior with the unmodified tyrosine peptide, and therefore a direct comparison of catalytic parameters for the respective substrates is not possible. For a qualitative estimate of substrate discrimination, a competition experiment was performed by incubating 2.5 nM sT4 with equimolar mixtures of the sTyr↓Arg and Tyr↓Arg substrates (1b and 3b) at five different substrate concentrations (50, 60, 70, 80, 90 μM each substrate; Figure S4 in the Supporting Information). The results indicated that sT4 exhibits about tenfold selectivity for the cleavage of the sulfated peptide. Consistent with its fluorescence profile, sT4 displayed only modest hydrolysis of the 2b pTyr↓Arg substrate, with kcat/KM = 5 ± 3 × 102M−1s−1, indicating a remarkable 200-fold selectivity in favor of sulfotyrosine over phosphotyrosine. Also, the enzyme showed no cleavage between pSer↓Arg even after overnight incubation (14 h) of the substrate 6b with a high concentration of the enzyme variant (0.5 μM). Finally, kcat/KM of sT4 for the hydrolysis of Glu↓Arg substrate (4b) was measured to be 3 ± 1 × 102M−1s−1, thus confirming only minor cross-reactivity with acidic residues. It is noteworthy that the selectivity of sT4 was not engineered at the expense of catalytic efficiency, since its kinetic parameters with its preferred sulfotyrosine substrate, 1b, are almost the same as WT OmpT with its preferred dibasic substrate 5b (Table 1). Although the reasons for sT4 discriminating between sulfotyrosine and phosphotyrosine are not known, a plausible explanation is that overall charge is important in substrate discrimination, as sulfotyrosine possesses an overall charge of −1 at neutral pH, while the overall charge on phosphotyrosine is −2. We are currently trying to crystallize the purified sT4 variant in complex with substrate analogue to identify the atomic interactions responsible for its selectivity, but that is beyond the scope of the current work. The ability to remodel OmpT activity to recognize selectively sulfotyrosine in P1 raises an intriguing issue regarding natural protease specificity. Could it be that certain natural proteases recognize post-translationally modified amino acids in biologically significant ways? There are at least two examples of proteases cleaving at post-translationally modified amino acids: Subtilisin BPN′ possesses appreciable activity towards phosphotyrosine-containing substrates,[11] and aminopeptidase Ey from chicken egg yolks can processively digest sulfotyrosine containing chemotactic peptides.[16] The successful engineering of a protease that selectively cleaves at sulfotyrosine residues in peptides marks the critical first step towards creating a practical enzyme useful for the detection of this interesting post-translational modification. Additional mutagenesis and screening of both targeted and random-error-prone mutant libraries of sT4 will be employed to achieve two important goals: 1) relax specificity for P1′-P3′ to accommodate all residues, especially acidic amino acids often found adjacent to sulfotyrosine and 2) increase the preference for sTyr/Tyr from tenfold to at least a 100-fold. These goals appear to be readily tractable given that OmpT exhibits relaxed selectivity for P1′ and P2′ and mutants[17] and our success in selectively altering the amino acid preference at P1′.[12]

IgG Fc domains that bind C1q but not effector Fc[gamma] receptors delineate the importance of complement-mediated effector functions

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.

Profiling human antibody responses by integrated single-cell analysis

Comprehensive characterization of the antigen-specific B cells induced during infections or following vaccination would facilitate the discovery of novel antibodies and inform how interventions shape protective humoral responses. The analysis of human B cells and their antibodies has been performed using flow cytometry to evaluate memory B cells and expanded plasmablasts, while microtechnologies have also provided a useful tool to examine plasmablasts/plasma cells after vaccination. Here we present an integrated analytical platform, using arrays of subnanoliter wells (nanowells), for constructing detailed profiles for human B cells comprising the immunophenotypes of these cells, the distribution of isotypes of the secreted antibodies, the specificity and relative affinity for defined antigens, and for a subset of cells, the genes encoding the heavy and light chains. The approach combines on-chip image cytometry, microengraving, and single-cell RT-PCR. Using clinical samples from HIV-infected subjects, we demonstrate that the method can identify antigen-specific neutralizing antibodies, is compatible with both plasmablasts/plasma cells and activated memory B cells, and is well-suited for characterizing the limited numbers of B cells isolated from tissue biopsies (e.g., colon biopsies). The technology should facilitate detailed analyses of human humoral responses for evaluating vaccines and their ability to raise protective antibody responses across multiple anatomical compartments.

Multi-copy genes that enhance the yield of mammalian G protein-coupled receptors in Escherichia coli.

Low yields of recombinant expression represent a major barrier to the physical characterization of membrane proteins. Here, we have identified genes that globally enhance the production of properly folded G protein-coupled receptors (GPCRs) in Escherichia coli. Libraries of bacterial chromosomal fragments were screened using two separate systems that monitor: (i) elevated fluorescence conferred by enhanced expression of GPCR-GFP fusions and (ii) increased binding of fluorescent ligand in cells producing more active receptor. Three multi-copy hits were isolated by both methods: nagD, encoding the ribonucleotide phosphatase NagD; a fragment of nlpD, encoding a truncation of the predicted lipoprotein NlpD, and the three-gene cluster ptsN-yhbJ-npr, encoding three proteins of the nitrogen phosphotransferase system. Expression of these genes resulted in a 3- to 10-fold increase in the yields of different mammalian GPCRs. Our data is consistent with the hypothesis that the expression of these genes may serve to maintain the integrity of the bacterial periplasm and to provide a favorable environment for proper membrane protein folding, possibly by inducing a fine-tuned stress response and/or via modifying the composition of the bacterial cell envelope.