Local fitness landscape of the green fluorescent protein.

Fitness landscapes depict how genotypes manifest at the phenotypic level and form the basis of our understanding of many areas of biology, yet their properties remain elusive. Previous studies have analysed specific genes, often using their function as a proxy for fitness, experimentally assessing the effect on function of single mutations and their combinations in a specific sequence or in different sequences. However, systematic high-throughput studies of the local fitness landscape of an entire protein have not yet been reported. Here we visualize an extensive region of the local fitness landscape of the green fluorescent protein from Aequorea victoria (avGFP) by measuring the native function (fluorescence) of tens of thousands of derivative genotypes of avGFP. We show that the fitness landscape of avGFP is narrow, with 3/4 of the derivatives with a single mutation showing reduced fluorescence and half of the derivatives with four mutations being completely non-fluorescent. The narrowness is enhanced by epistasis, which was detected in up to 30% of genotypes with multiple mutations and mostly occurred through the cumulative effect of slightly deleterious mutations causing a threshold-like decrease in protein stability and a concomitant loss of fluorescence. A model of orthologous sequence divergence spanning hundreds of millions of years predicted the extent of epistasis in our data, indicating congruence between the fitness landscape properties at the local and global scales. The characterization of the local fitness landscape of avGFP has important implications for several fields including molecular evolution, population genetics and protein design.

FLIM of NAD(P)H in Lymphatic Nodes Resolves T-Cell Immune Response to the Tumor.

Assessment of T-cell response to the tumor is important for diagnosis of the disease and monitoring of therapeutic efficacy. For this, new non-destructive label-free methods are required. Fluorescence lifetime imaging (FLIM) of metabolic coenzymes is a promising innovative technology for the assessment of the functional status of cells. The purpose of this work was to test whether FLIM can resolve metabolic alterations that accompany T-cell reactivation to the tumors. The study was carried out on C57Bl/6 FoxP3-EGFP mice bearing B16F0 melanoma. Autofluorescence of the immune cells in fresh lymphatic nodes (LNs) was investigated. It was found that fluorescence lifetime parameters of nicotinamide adenine dinucleotide (phosphate) NAD(P)H are sensitive to tumor development. Effector T-cells in the LNs displayed higher contribution of free NADH, the form associated with glycolysis, in all tumors and the presence of protein-bound NADPH, associated with biosynthetic processes, in the tumors of large size. Flow cytometry showed that the changes in the NADH fraction of the effector T-cells correlated with their activation, while changes in NADPH correlated with cell proliferation. In conclusion, FLIM of NAD(P)H in fresh lymphoid tissue is a powerful tool for assessing the immune response to tumor development.

Deciphering Repertoire of B16 Melanoma Reactive TCRs by Immunization, In Vitro Restimulation and Sequencing of IFNγ-Secreting T Cells.

Recent advances in cancer immunotherapy have great promise for the treatment of solid tumors. One of the key limiting factors that hamper the decoding of physiological responses to these therapies is the inability to distinguish between specific and nonspecific responses. The identification of tumor-specific lymphocytes is also the most challenging step in cancer cell therapies such as adoptive cell transfer and T cell receptor (TCR) cloning. Here, we have elaborated a protocol for the identification of tumor-specific T lymphocytes and the deciphering of their repertoires. B16 melanoma engraftment following anti-PD1 checkpoint therapy provides better antitumor immunity compared to repetitive immunization with heat-shocked tumor cells. We have also revealed that the most error-prone part of dendritic cell (DC) generation, i.e., their maturation step, can be omitted if DCs are cultured at a sufficiently high density. Using this optimized protocol, we have achieved a robust IFNγ response to B16F0 antigens, but only within CD4+ T helper cells. A comparison of the repertoires of IFNγ-positive and -negative cells shows a prominent enrichment of certain clones with putative tumor specificity among the IFNγ+ fraction. In summary, our optimized protocol and the data provided here will aid in the acquisition of broad statistical data and the creation of a meaningful database of B16-specific TCRs.

Expression of EMT-Related Genes in Hybrid E/M Colorectal Cancer Cells Determines Fibroblast Activation and Collagen Remodeling.

Collagen, the main non-cellular component of the extracellular matrix (ECM), is profoundly reorganized during tumorigenesis and has a strong impact on tumor behavior. The main source of collagen in tumors is cancer-associated fibroblasts. Cancer cells can also participate in the synthesis of ECM; however, the contribution of both types of cells to collagen rearrangements during the tumor progression is far from being clear. Here, we investigated the processes of collagen biosynthesis and remodeling in parallel with the transcriptome changes during cancer cells and fibroblasts interactions. Combining immunofluorescence, RNA sequencing, and second harmonic generation microscopy, we have explored the relationships between the ratio of epithelial (E) and mesenchymal (M) components of hybrid E/M cancer cells, their ability to activate fibroblasts, and the contributions of both cell types to collagen remodeling. To this end, we studied (i) co-cultures of colorectal cancer cells and normal fibroblasts in a collagen matrix, (ii) patient-derived cancer-associated fibroblasts, and (iii) mouse xenograft models. We found that the activation of normal fibroblasts that form dense collagen networks consisting of large, highly oriented fibers depends on the difference in E/M ratio in the cancer cells. The more-epithelial cells activate the fibroblasts more strongly, which correlates with a dense and highly ordered collagen structure in tumors in vivo. The more-mesenchymal cells activate the fibroblasts to a lesser degree; on the other hand, this cell line has a higher innate collagen remodeling capacity. Normal fibroblasts activated by cancer cells contribute to the organization of the extracellular matrix in a way that is favorable for migratory potency. At the same time, in co-culture with epithelial cancer cells, the contribution of fibroblasts to the reorganization of ECM is more pronounced. Therefore, one can expect that targeting the ability of epithelial cancer cells to activate normal fibroblasts may provide a new anticancer therapeutic strategy.

Quantitative profiling of immune repertoires for minor lymphocyte counts using unique molecular identifiers.

Emerging high-throughput sequencing methods for the analyses of complex structure of TCR and BCR repertoires give a powerful impulse to adaptive immunity studies. However, there are still essential technical obstacles for performing a truly quantitative analysis. Specifically, it remains challenging to obtain comprehensive information on the clonal composition of small lymphocyte populations, such as Ag-specific, functional, or tissue-resident cell subsets isolated by sorting, microdissection, or fine needle aspirates. In this study, we report a robust approach based on unique molecular identifiers that allows profiling Ag receptors for several hundred to thousand lymphocytes while preserving qualitative and quantitative information on clonal composition of the sample. We also describe several general features regarding the data analysis with unique molecular identifiers that are critical for accurate counting of starting molecules in high-throughput sequencing applications.

Soluble OX40L favors tumor rejection in CT26 colon carcinoma model.

OX40 receptor-expressing regulatory T cells (Tregs) populate tumors and suppress a variety of immune cells, posing a major obstacle for cancer immunotherapy. Different ways to functionally inactivate Tregs by triggering OX40 receptor have been suggested, including anti-OX40 antibodies and Fc:OX40L fusion proteins. To investigate whether the soluble extracellular domain of OX40L (OX40Lexo) is sufficient to enhance antitumor immune response, we generated an OX40Lexo-expressing CT26 colon carcinoma cell line and studied its tumorigenicity in immunocompetent BALB/c and T cell deficient nu/nu mice. We found that soluble OX40L expressed in CT26 colon carcinoma favors the induction of an antitumor response which is not limited just to cells co-expressing EGFP as an antigenic determinant, but also eliminates CT26 cells expressing another fluorescent protein, KillerRed. Tumor rejection required the presence of T lymphocytes, as indicated by the unhampered tumor growth in nu/nu mice. Subsequent re-challenge of tumor-free BALB/c mice with CT26 EGFP cells resulted in no tumor growth, which is indicative of the formation of immunological memory. Adoptive transfer of splenocytes from mice that successfully rejected CT26 OX40Lexo EGFP tumors to naïve mice conferred 100% resistance to subsequent challenge with the CT26 EGFP tumor.

Green fluorescent protein with anionic tryptophan-based chromophore and long fluorescence lifetime.

Spectral diversity of fluorescent proteins, crucial for multiparameter imaging, is based mainly on chemical diversity of their chromophores. Recently we have reported, to our knowledge, a new green fluorescent protein WasCFP-the first fluorescent protein with a tryptophan-based chromophore in the anionic state. However, only a small portion of WasCFP molecules exists in the anionic state at physiological conditions. In this study we report on an improved variant of WasCFP, named NowGFP, with the anionic form dominating at 37°C and neutral pH. It is 30% brighter than enhanced green fluorescent protein (EGFP) and exhibits a fluorescence lifetime of 5.1 ns. We demonstrated that signals of NowGFP and EGFP can be clearly distinguished by fluorescence lifetime in various models, including mammalian cells, mouse tumor xenograft, and Drosophila larvae. NowGFP thus provides an additional channel for multiparameter fluorescence lifetime imaging microscopy of green fluorescent proteins.

Point mutations in dimerization motifs of the transmembrane domain stabilize active or inactive state of the EphA2 receptor tyrosine kinase.

The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L(535)X3G(539)X2A(542)X3V(546)X2L(549) rather than through the alternative glycine zipper motif A(536)X3G(540)X3G(544) (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr(588) and/or Tyr(594)) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.

Pairing of T-cell receptor chains via emulsion PCR.

Our ability to analyze adaptive immunity and engineer its activity has long been constrained by our limited ability to identify native pairs of heavy-light antibody chains and alpha-beta T-cell receptor (TCR) chains--both of which comprise coupled "halves of a key", collectively capable of recognizing specific antigens. Here, we report a cell-based emulsion RT-PCR approach that allows the selective fusion of the native pairs of amplified TCR alpha and beta chain genes for complex samples. A new type of PCR suppression technique was developed that makes it possible to amplify the fused library with minimal noise for subsequent analysis by high-throughput paired-end Illumina sequencing. With this technique, single analysis of a complex blood sample allows identification of multiple native TCR chain pairs. This approach may be extended to identify native antibody chain pairs and, more generally, pairs of mRNA molecules that are coexpressed in the same living cells.

Local Enrichment with Convergence of Enriched T-Cell Clones Are Hallmarks of Effective Peptide Vaccination against B16 Melanoma.

BACKGROUND: Some peptide anticancer vaccines elicit a strong T-cell memory response but fail to suppress tumor growth. To gain insight into tumor resistance, we compared two peptide vaccines, p20 and p30, against B16 melanoma, with both exhibiting good in vitro T-cell responses but different tumor suppression abilities. METHODS: We compared activation markers and repertoires of T-lymphocytes from tumor-draining (dLN) and non-draining (ndLN) lymph nodes for the two peptide vaccines. RESULTS: We showed that the p30 vaccine had better tumor control as opposed to p20. p20 vaccine induced better in vitro T-cell responsiveness but failed to suppress tumor growth. Efficient antitumor vaccination is associated with a higher clonality of cytotoxic T-cells (CTLs) in dLNs compared with ndLNs and the convergence of most of the enriched clones. With the inefficient p20 vaccine, the most expanded and converged were clones of the bystander T-cells without an LN preference. CONCLUSIONS: Here, we show that the clonality and convergence of the T-cell response are the hallmarks of efficient antitumor vaccination. The high individual and methodological dependencies of these parameters can be avoided by comparing dLNs and ndLNs.

Light-induced blockage of cell division with a chromatin-targeted phototoxic fluorescent protein.

Proteins of the GFP (green fluorescent protein) family are widely used as passive reporters for live cell imaging. In the present study we used H2B (histone H2B)-tKR (tandem KillerRed) as an active tool to affect cell division with light. We demonstrated that H2B-tKR-expressing cells behave normally in the dark, but transiently cease proliferation following green-light illumination. Complete light-induced blockage of cell division for approx. 24 h was observed in cultured mammalian cells that were either transiently or stably transfected with H2B-tKR. Illuminated cells then returned to normal division rate. XRCC1 (X-ray cross complementing factor 1) showed immediate redistribution in the illuminated nuclei of H2B-tKR-expressing cells, indicating massive light-induced damage of genomic DNA. Notably, nondisjunction of chromosomes was observed for cells that were illuminated during metaphase. In transgenic Xenopus embryos expressing H2B-tKR under the control of tissue-specific promoters, we observed clear retardation of the development of these tissues in green-light-illuminated tadpoles. We believe that H2B-tKR represents a novel optogenetic tool, which can be used to study mitosis and meiosis progression per se, as well as to investigate the roles of specific cell populations in development, regeneration and carcinogenesis in vivo.

Pinpointing the tumor-specific T cells via TCR clusters.

Adoptive cell transfer (ACT) is a promising approach to cancer immunotherapy, but its efficiency fundamentally depends on the extent of tumor-specific T cell enrichment within the graft. This can be estimated via activation with identifiable neoantigens, tumor-associated antigens (TAAs), or living or lysed tumor cells, but these approaches remain laborious, time-consuming, and functionally limited, hampering clinical development of ACT. Here, we demonstrate that homology cluster analysis of T cell receptor (TCR) repertoires efficiently identifies tumor-reactive TCRs allowing to: (1) detect their presence within the pool of tumor-infiltrating lymphocytes (TILs); (2) optimize TIL culturing conditions, with IL-2low/IL-21/anti-PD-1 combination showing increased efficiency; (3) investigate surface marker-based enrichment for tumor-targeting T cells in freshly isolated TILs (enrichment confirmed for CD4+ and CD8+ PD-1+/CD39+ subsets), or re-stimulated TILs (informs on enrichment in 4-1BB-sorted cells). We believe that this approach to the rapid assessment of tumor-specific TCR enrichment should accelerate T cell therapy development.

Bioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers.

Screening drug candidates for their affinity and selectivity for a certain binding site is a crucial step in developing targeted therapy. Here, we created a screening assay for receptor binding that can be easily scaled up and automated for the high throughput screening of Kv channel blockers. It is based on the expression of the KcsA-Kv1 hybrid channel tagged with a fluorescent protein in the E. coli membrane. In order to make this channel accessible for the soluble compounds, E. coli were transformed into spheroplasts by disruption of the cellular peptidoglycan envelope. The assay was evaluated using a hybrid KcsA-Kv1.3 potassium channel tagged with a red fluorescent protein (TagRFP). The binding of Kv1.3 channel blockers was measured by flow cytometry either by using their fluorescent conjugates or by determining the ability of unconjugated compounds to displace fluorescently labeled blockers with a known affinity. A fraction of the occupied receptor was calculated with a dedicated pipeline available as a Jupyter notebook. Measured binding constants for agitoxin-2, charybdotoxin and kaliotoxin were in firm agreement with the earlier published data. By using a mid-range flow cytometer with manual sample handling, we measured and analyzed up to ten titration curves (eight data points each) in one day. Finally, we considered possibilities for multiplexing, scaling and automation of the assay.

B cells, plasma cells and antibody repertoires in the tumour microenvironment.

Recent data show that B cells and plasma cells located in tumours or in tumour-draining lymph nodes can have important roles in shaping antitumour immune responses. In tumour-associated tertiary lymphoid structures, T cells and B cells interact and undergo cooperative selection, specialization and clonal expansion. Importantly, B cells can present cognate tumour-derived antigens to T cells, with the functional consequences of such interactions being shaped by the B cell phenotype. Furthermore, the isotype and specificity of the antibodies produced by plasma cells can drive distinct immune responses. Here we summarize our current knowledge of the roles of B cells and antibodies in the tumour microenvironment. Moreover, we discuss the potential of using immunoglobulin repertoires as a source of tumour-specific receptors for immunotherapy or as biomarkers to predict the efficacy of immunotherapeutic interventions.

RNA-Seq-Based TCR Profiling Reveals Persistently Increased Intratumoral Clonality in Responders to Anti-PD-1 Therapy.

Substantial effort is being invested in the search for peripheral or intratumoral T cell receptor (TCR) repertoire features that could predict the response to immunotherapy. Here we demonstrate the utility of MiXCR software for TCR and immunoglobulin repertoire extraction from RNA-Seq data obtained from sorted tumor-infiltrating T and B cells. We use this approach to extract TCR repertoires from RNA-Seq data obtained from sorted tumor-infiltrating CD4+ and CD8+ T cells in an HKP1 (KrasG12Dp53-/-) syngeneic mouse model of lung cancer after anti-PD-1 treatment. For both subsets, we demonstrate decreased TCR diversity in response to therapy. At a later time point, repertoire diversity is restored in progressing disease but remains decreased in responders to therapy in both CD4+ and CD8+ subsets. These observations complement previous studies and suggest that stably increased intratumoral CD4+ and CD8+ T cell clonality after anti-PD-1/PD-L1 therapy could serve as a predictor of long-term response.

Measuring Intratumoral Heterogeneity of Immune Repertoires.

There is considerable clinical and fundamental value in measuring the clonal heterogeneity of T and B cell expansions in tumors and tumor-associated lymphoid structures-along with the associated heterogeneity of the tumor neoantigen landscape-but such analyses remain challenging to perform. Here, we propose a straightforward approach to analyze the heterogeneity of immune repertoires between different tissue sections in a quantitative and controlled way, based on a beta-binomial noise model trained on control replicates obtained at the level of single-cell suspensions. This approach allows to identify local clonal expansions with high accuracy. We reveal in situ proliferation of clonal T cells in a mouse model of melanoma, and analyze heterogeneity of immunoglobulin repertoires between sections of a metastatically-infiltrated lymph node in human melanoma and primary human colon tumor. On the latter example, we demonstrate the importance of training the noise model on datasets with depth and content that is comparable to the samples being studied. Altogether, we describe here the crucial basic instrumentarium needed to facilitate proper experimental setup planning in the rapidly evolving field of intratumoral immune repertoires, from the wet lab to bioinformatics analysis.

Data supporting that adipose-derived mesenchymal stem/stromal cells express angiotensin II receptors in situ and in vitro.

This article contains results of analyses of angiotensin II receptors expression in human adipose tissue and stem/stromal cells isolated from adipose tissue. We also provide here data regarding the effect of angiotensin II on intracellular calcium mobilization in adipose tissue derived stem/stromal cells (ADSCs). Discussion of the data can be found in (Sysoeva et al., 2017) [1].

Comparative Analysis of B-Cell Receptor Repertoires Induced by Live Yellow Fever Vaccine in Young and Middle-Age Donors.

Age-related changes can significantly alter the state of adaptive immune system and often lead to attenuated response to novel pathogens and vaccination. In present study we employed 5'RACE UMI-based full length and nearly error-free immunoglobulin profiling to compare plasma cell antibody repertoires in young (19-26 years) and middle-age (45-58 years) individuals vaccinated with a live yellow fever vaccine, modeling a newly encountered pathogen. Our analysis has revealed age-related differences in the responding antibody repertoire ranging from distinct IGH CDR3 repertoire properties to differences in somatic hypermutation intensity and efficiency and antibody lineage tree structure. Overall, our findings suggest that younger individuals respond with a more diverse antibody repertoire and employ a more efficient somatic hypermutation process than elder individuals in response to a newly encountered pathogen.

Molecular Mechanisms of Immunomodulation Properties of Mesenchymal Stromal Cells: A New Insight into the Role of ICAM-1.

Mesenchymal stromal cells (MSC) control excessive inflammation and create a microenvironment for tissue repair protecting from chronic inflammation and tissue fibrosis. We examined the molecular mechanisms of MSC immunomodulatory function in mixed cultures of human adipose-derived MSC with lymphocytes. Our data show that MSC promote unstimulated lymphocyte survival potentially by an increase in antigen presentation. Under inflammatory conditions, mimicked by stimulation of TCR in lymphocytes, MSC suppress activation and proliferation of stimulated T cells. Immunosuppression is accompanied by downregulation of IL-2Rα that negatively affects the survival of activated T cells. MSC upregulate transcription of indolamine-2,3-dioxygenase (IDO) and inducible NO synthase (iNOS), which generate products negatively affecting T cell function. Both MSC and lymphocytes dramatically increase the surface ICAM-1 level in mixed cultures. Antibody-mediated blockage of surface ICAM-1 partially releases MSC-mediated immune suppression in vitro. Our data suggest that MSC have cell-intrinsic molecular programs depending on the inflammatory microenvironment. We speculate that MSC sense soluble factors and respond by surface ICAM-1 upregulation. ICAM-1 is involved in the control of T cell activation leading to immunosuppression or modest stimulation depending on the T cell status. Immunomodulation by MSC ranging from support of naive T cell survival to immunosuppression of activated T cells may affect the tissue microenvironment protecting from aberrant regeneration.

Local angiotensin II promotes adipogenic differentiation of human adipose tissue mesenchymal stem cells through type 2 angiotensin receptor.

Obesity is often associated with high systemic and local activity of renin-angiotensin system (RAS). Mesenchymal stem cells of adipose tissue are the main source of adipocytes. The aim of this study was to clarify how local RAS could control adipose differentiation of human adipose tissue derived mesenchymal stem cells (ADSCs). We examined the distribution of angiotensin receptor expressing cells in human adipose tissue and found that type 1 and type 2 receptors are co-expressed in its stromal compartment, which is known to contain mesenchymal stem cells. To study the expression of receptors specifically in ADSCs we have isolated them from adipose tissue. Up to 99% of cultured ADSCs expressed angiotensin II (AngII) receptor type 1 (AT1). Using the analysis of Ca2+ mobilization in single cells we found that only 5.2±2.7% of ADSCs specifically respond to serial Ang II applications via AT1 receptor and expressed this receptor constantly. This AT1const ADSCs subpopulation exhibited increased adipose competency, which was triggered by endogenous AngII. Inhibitory and expression analyses showed that AT1const ADSCs highly co-express AngII type 2 receptor (AT2), which was responsible for increased adipose competency of this ADSC subpopulation.