Adhering interacting cells to two opposing coverslips allows super-resolution imaging of cell-cell interfaces.

Cell-cell interfaces convey mechanical and chemical information in multicellular systems. Microscopy has revealed intricate structure of such interfaces, yet typically with limited resolution due to diffraction and unfavourable orthogonal orientation of the interface to the coverslip. We present a simple and robust way to align cell-cell interfaces in parallel to the coverslip by adhering the interacting cells to two opposing coverslips. We demonstrate high-quality diffraction-limited and super-resolution imaging of interfaces (immune-synapses) between fixed and live CD8+ T-cells and either antigen presenting cells or melanoma cells. Imaging methods include bright-field, confocal, STED, dSTORM, SOFI, SRRF and large-scale tiled images. The low background, lack of aberrations and enhanced spatial stability of our method relative to existing cell-trapping techniques allow use of these methods. We expect that the simplicity and wide-compatibility of our approach will allow its wide dissemination for super-resolving the intricate structure and molecular organization in a variety of cell-cell interfaces.

Maturation of T and B Lymphocytes in the Assessment of the Immune Status in COVID-19 Patients.

Cell response to novel coronavirus disease 19 (COVID-19) is currently a widely researched topic. The assessment of leukocytes population and the maturation of both B and T lymphocytes may be important in characterizing the immunological profile of COVID-19 patients. The aim of the present study was to evaluate maturation of B and T cells in COVID-19 patients with interstitial lesions on chest X-ray (COVID-19 X-ray (+)), without changes on X-ray (COVID-19 X-ray (-)) and in healthy control. The study group consisted of 23 patients divided on two groups: COVID-19 X-ray (+) n = 14 and COVID-19 X-ray (-) n = 9 and control n = 20. The flow cytometry method was performed. We observed a significantly higher percentage of plasmablasts and lower CD4+ lymphocytes in COVID-19 X-ray (+) patients than in COVID-19 X-ray (-) and control. In the COVID-19 X-ray (+) patients, there was a lower proportion of effector CD4+ T cells, naïve CD8+ T cells and higher central memory CD4+ cells and effector CD8+ T cells than control. The above results showed that the assessment of selected cells of B and T lymphocytes by flow cytometry can distinguish patients with COVID-19 and differentiate patients with and without changes on chest X-ray.

Post-hoc assessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms.

Biologics have the potential to induce an immune response when used therapeutically. A number of in vitro assays are currently used preclinically to predict the risk of immunogenicity, but the validation of these preclinical tools suffers from the relatively small number of accessible immunogenic molecules and the limited understanding of the mechanisms underlying the immunogenicity of biologics. Here, we present the post-hoc analysis of three monoclonal antibodies with high immunogenicity in the clinic. Two of the three antibodies elicited a CD4 T cell proliferative response in multiple donors in a peripheral blood mononuclear cell assay, but required different experimental conditions to induce these responses. The third antibody did not trigger any T cell response in this assay. These distinct capacities to promote CD4 T cell responses in vitro were mirrored by different capacities to stimulate innate immune cells. Only one of the three antibodies was capable of inducing human dendritic cell (DC) maturation; the second antibody promoted monocyte activation while the third one did not induce any innate cell activation in vitro. All three antibodies exhibited a moderate to high internalization by human DCs and MHC-associated peptide proteomics analysis revealed the presence of potential T cell epitopes that were confirmed by a T-cell proliferation assay. Collectively, these findings highlight the existence of distinct immune stimulatory mechanisms for immunogenic antibodies. These findings have implications for the preclinical immunogenicity risk assessment of biologics.

Quantitative assessment of cell population diversity in single-cell landscapes.

Single-cell RNA sequencing (scRNA-seq) has become a powerful tool for the systematic investigation of cellular diversity. As a number of computational tools have been developed to identify and visualize cell populations within a single scRNA-seq dataset, there is a need for methods to quantitatively and statistically define proportional shifts in cell population structures across datasets, such as expansion or shrinkage or emergence or disappearance of cell populations. Here we present sc-UniFrac, a framework to statistically quantify compositional diversity in cell populations between single-cell transcriptome landscapes. sc-UniFrac enables sensitive and robust quantification in simulated and experimental datasets in terms of both population identity and quantity. We have demonstrated the utility of sc-UniFrac in multiple applications, including assessment of biological and technical replicates, classification of tissue phenotypes and regional specification, identification and definition of altered cell infiltrates in tumorigenesis, and benchmarking batch-correction tools. sc-UniFrac provides a framework for quantifying diversity or alterations in cell populations across conditions and has broad utility for gaining insight into tissue-level perturbations at the single-cell resolution.

Assessment of Humoral, Innate, and T-Cell Immune Responses to Adeno-Associated Virus Vectors.

Adeno-associated virus (AAV)-based gene therapy is being applied to treat a wide array of diseases. Preexisting host immune responses to AAV and immune responses elicited by AAV vector administration remain a problem that needs to be further studied. Here we present a series of protocols to assess immune responses before and after AAV vector administration that are applicable to multiple animal models and phase 1 clinical trials. More specifically, they may be use to evaluate (1) the humoral immune response, through levels of AAV-neutralizing and binding antibodies; (2) the innate immune response, through the acute induction of inflammatory cytokines; and (3) the T-cell immune response, through the activation of transgene- and vector-specific CD8+ and CD4+ T cells.

Heterogeneity assessment of functional T cell avidity.

The potency of cellular immune responses strongly depends on T cell avidity to antigen. Yet, functional avidity measurements are rarely performed in patients, mainly due to the technical challenges of characterizing heterogeneous T cells. The mean functional T cell avidity can be determined by the IFN-γ Elispot assay, with titrated amounts of peptide. Using this assay, we developed a method revealing the heterogeneity of functional avidity, represented by the steepness/hillslope of the peptide titration curve, documented by proof of principle experiments and mathematical modeling. Our data show that not only natural polyclonal CD8 T cell populations from cancer patients, but also monoclonal T cells differ strongly in their heterogeneity of functional avidity. Interestingly, clones and polyclonal cells displayed comparable ranges of heterogeneity. We conclude that besides the mean functional avidity, it is feasible and useful to determine its heterogeneity (hillslope) for characterizing T cell responses in basic research and patient investigation.

Assessment of Antigen-Specific Cellular Immunogenicity Using Intracellular Cytokine Staining, ELISpot, and Culture Supernatants.

Quantification of cytokine production by CD4 and CD8 T cells after in vitro recall stimulation with the immunizing antigen is a powerful approach to characterize the cellular immune responses to immunization. Here we describe three complementary methods for such quantification including flow cytometric analysis of cytokine production by intracellular staining, ELISpot determination of the numbers of cytokine-producing cells, and generation of secreted cytokines and chemokines in culture supernatants for analysis by ELISA and/or cytometric bead arrays.

Quantitative assessment of the functional plasticity of memory CD8(+) T cells.

While the functional plasticity of memory CD4(+) T cells has been studied extensively, less is known about this property in memory CD8(+) T cells. Here, we report the direct measurement of plasticity by paired daughter analysis of effector and memory OT-I CD8(+) T cells primed in vivo with ovalbumin. Naïve, effector, and memory OT-I cells were isolated and activated in single-cell culture; then, after the first division, their daughter cells were transferred to new cultures with and without IL-4; expression of IFN-γ and IL-4 mRNAs was measured 5 days later in the resultant subclones. Approximately 40% of clonogenic memory CD8(+) T cells were bipotential in this assay, giving rise to an IL-4(-) subclone in the absence of IL-4 and an IL-4(+) subclone in the presence of IL-4. The frequency of bipotential cells was lower among memory cells than naïve cells but markedly higher than among 8-day effectors. Separation based on high or low expression of CD62L, CD122, CD127, or Ly6C did not identify a phenotypic marker of the bipotential cells. Functional plasticity in memory CD8(+) T-cell populations can therefore reflect modulation at the level of a single memory cell and its progeny.

Quantitative TCR:pMHC Dissociation Rate Assessment by NTAmers Reveals Antimelanoma T Cell Repertoires Enriched for High Functional Competence.

Experimental models demonstrated that therapeutic induction of CD8 T cell responses may offer protection against tumors or infectious diseases providing that T cells have sufficiently high TCR/CD8:pMHC avidity for efficient Ag recognition and consequently strong immune functions. However, comprehensive characterization of TCR/CD8:pMHC avidity in clinically relevant situations has remained elusive. In this study, using the novel NTA-His tag-containing multimer technology, we quantified the TCR:pMHC dissociation rates (koff) of tumor-specific vaccine-induced CD8 T cell clones (n = 139) derived from seven melanoma patients vaccinated with IFA, CpG, and the native/EAA or analog/ELA Melan-A(MART-1)(26-35) peptide, binding with low or high affinity to MHC, respectively. We observed substantial correlations between koff and Ca(2+) mobilization (p = 0.016) and target cell recognition (p < 0.0001), with the latter independently of the T cell differentiation state. Our strategy was successful in demonstrating that the type of peptide impacted on TCR/CD8:pMHC avidity, as tumor-reactive T cell clones derived from patients vaccinated with the low-affinity (native) peptide expressed slower koff rates than those derived from patients vaccinated with the high-affinity (analog) peptide (p < 0.0001). Furthermore, we observed that the low-affinity peptide promoted the selective differentiation of tumor-specific T cells bearing TCRs with high TCR/CD8:pMHC avidity (p < 0.0001). Altogether, TCR:pMHC interaction kinetics correlated strongly with T cell functions. Our study demonstrates the feasibility and usefulness of TCR/CD8:pMHC avidity assessment by NTA-His tag-containing multimers of naturally occurring polyclonal T cell responses, which represents a strong asset for the development of immunotherapy.

Assessment of tissue FoxP3+, CD4+ and CD8+ T-cells in active and stable nonsegmental vitiligo.

BACKGROUND: The exact etiology of vitiligo remains obscure. Studies have indicated a role for cellular immunity in the pathogenesis of vitiligo. The aim of this study is to assess tissue FoxP3+ natural regulatory T-cells (Tregs), as well as CD4+ and CD8+ T-cells in active vs. stable nonsegmental vitiligo. MATERIALS AND METHODS: Immunohistochemical double-staining for expression of CD4+ and CD8+ T-cells with immunostaining for expression of FoxP3 in lesional, marginal, and nonlesional skin of nonsegmental vitiligo was used to evaluate the abundance of Tregs among CD4+ and CD8+ T-cells in active and stable cases of vitiligo. RESULTS: A significant increase in the number of CD4+ and CD8+ T-cells and a highly significant reduction in the number of FoxP3-expressing Tregs were detected in marginal skin in both stable and active vitiligo cases. FoxP3+ cells were decreased in tissue of patients with vitiligo compared with healthy controls. The number of CD8+ T-cells was increased in the epidermis and dermoepidermal junction (DEJ) in comparison with the number of CD4+ T-cells. Tregs were mostly present at the DEJ. CONCLUSION: The reduction in the number of FoxP3+ cells in the marginal skin suggests that this is the site where regulatory activity is needed to suppress the activity of helper and cytotoxic T-cells that are actively contributing to depigmentation.

Leukoreduction system chambers are an efficient, valid, and economic source of functional monocyte-derived dendritic cells and lymphocytes.

The demand for human monocyte-derived dendritic cells (moDCs), as well as for primary human B and T lymphocytes for immunological research purposes has been increased in recent years. Classically, these monocytes are isolated from blood, leukapheresis products or buffy coats of healthy donors by plastic adherence of peripheral blood mononuclear cells (PBMCs), followed by stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, while lymphocytes are usually isolated from the non-adherent fraction (NAF) by magnetic cell sorting. However, donor-blood is a limited resource and not every blood bank offers leukapheresis products or buffy coats for laboratory use. Additionally, a leukapheresis is very expensive and also the generation/isolation of cells is time- and cost-intensive. To overcome some of these obstacles, we evaluated if low-cost leukoreduction system chambers (LRSCs), which arise after routine donor plateletpheresis procedures, and are usually discarded, would be an alternative and appropriate source of PBMCs to generate moDCs and to isolate lymphocytes. By analyzing the number and phenotype of immature and mature dendritic cells (DCs), as well as of B and T lymphocytes derived from LRSCs, we found all cells to be of high quantity and quality. Further investigations on DCs comprising transwell migration assays, allogeneic mixed lymphocyte reactions (MLR), cytokine secretion assays, and cytotoxic T cell induction assays revealed high migratory, as well as stimulatory capacity of these cells. In addition, DCs and T cells were efficiently electroporated with mRNA and showed characteristic cytokine production after co-culture, demonstrating LRSCs as an efficient, valid, and economic source for generation of moDCs and lymphocytes for research purposes.

Assessment of CD4(+) and CD8 (+) T cell responses using MHC class I and II tetramers.

The low frequency of T cells specific for given antigens makes the study of antigen-specific T cell responses difficult. The development of MHC class I and II tetramer staining techniques allows precise quantification and tracking of antigen-specific CD8(+) and CD4(+) T cell responses. Here, we describe a protocol for MHC class I and II tetramer staining of mouse T cells isolated from various tissues of mice infected with lymphocytic choriomeningitis virus (LCMV) or with murine cytomegalovirus (MCMV).

Assessment of immunosuppressive activity of human mesenchymal stem cells using murine antigen specific CD4 and CD8 T cells in vitro.

INTRODUCTION: Mesenchymal stem cells (MSCs) have immunosuppressive activity. They do not induce allospecific T cell responses, making them promising tools for reducing the severity of graft versus host disease (GVHD) as well as treating various immune diseases. Currently, there is a need in the MSC field to develop a robust in vitro bioassay which can characterize the immunosuppressive function of MSCs. METHODS: Murine clonal CD4 and CD8 T cells were stimulated with cognate peptide antigen and antigen presenting cells (APCs) in the absence or presence of human MSCs, different aspects of T cell activation were monitored and analyzed using flow cytometry, real time RT-PCR and cytokine measurement. RESULTS: Human MSCs (hMSCs) can alter multiple aspects of murine T cell activation induced by stimulation with specific antigen, including: reduced proliferation, inhibited or stimulated cell surface marker expression (CD25, CD69, CD44 and CD62L), inhibited mRNA expression of transcription factors (T-bet and GATA-3) and decreased cytokine expression (interferon-gamma, interleukin-10). Disappearance of activation-induced cluster formation and decreased apoptosis of CD8 T cells were also observed. Moreover, the effects are specific to MSCs; incubating the T cells with non-MSC control cell lines had no effect on T cell proliferation and activation. CONCLUSIONS: Clonal murine T cells can be used to measure, characterize, and quantify the in vitro immunosuppressive activity of human MSCs, representing a promising approach to improve bioassays for immunosuppression.

A critical assessment for the value of markers to gate-out undesired events in HLA-peptide multimer staining protocols.

BACKGROUND: The introduction of antibody markers to identify undesired cell populations in flow-cytometry based assays, so called DUMP channel markers, has become a practice in an increasing number of labs performing HLA-peptide multimer assays. However, the impact of the introduction of a DUMP channel in multimer assays has so far not been systematically investigated across a broad variety of protocols. METHODS: The Cancer Research Institute's Cancer Immunotherapy Consortium (CRI-CIC) conducted a multimer proficiency panel with a specific focus on the impact of DUMP channel use. The panel design allowed individual laboratories to use their own protocol for thawing, staining, gating, and data analysis. Each experiment was performed twice and in parallel, with and without the application of a dump channel strategy. RESULTS: The introduction of a DUMP channel is an effective measure to reduce the amount of non-specific MULTIMER binding to T cells. Beneficial effects for the use of a DUMP channel were observed across a wide range of individual laboratories and for all tested donor-antigen combinations. In 48% of experiments we observed a reduction of the background MULTIMER-binding. In this subgroup of experiments the median background reduction observed after introduction of a DUMP channel was 0.053%. CONCLUSIONS: We conclude that appropriate use of a DUMP channel can significantly reduce background staining across a large fraction of protocols and improve the ability to accurately detect and quantify the frequency of antigen-specific T cells by multimer reagents. Thus, use of a DUMP channel may become crucial for detecting low frequency antigen-specific immune responses. Further recommendations on assay performance and data presentation guidelines for publication of MULTIMER experimental data are provided.

Flow cytometric assessment of antigen-specific proliferation in peripheral chicken T cells by CFSE dilution.

Carboxyfluorescein succinimidyl ester (CFSE) dilution is a well established method for analysis of dividing cells by flow cytometry. In other species the method has been extensively used in the study of antigen-specific T cells. The purpose of this study was to apply the method to chicken peripheral mononuclear blood cells (PBMC) and to evaluate and optimize its performance in relation to detection of vaccine-induced chicken T cells specific for Newcastle disease virus (NDV). The method was based on analysis of CFSE dilution upon ex vivo recall stimulation with whole vaccine antigen. Analysis of proliferation was combined with the use of monoclonal antibodies directed against the lymphocyte surface markers CD4 and CD8 in order to phenotype the responding cells. Problems with nonspecific background proliferation especially in the CD8 compartment were significantly reduced by replacing medium containing fetal calf serum with serum-free medium. It was rendered probable that antigen-specific cellular immunity can be assessed by this method as NDV-vaccinated chickens showed a significantly higher proliferative capacity than age-matched naïve controls. Furthermore it was shown that the recall stimulation lead to a proliferative response in T cells expressing αß-type TCRs but also those expressing the γδ-type. In summary, the method was found challenging but nevertheless useful to quantify the proliferative response of chicken antigen-specific T cells. Further investigations though, are needed in order to prove what cell subsets are true antigen-specific responders and what cells are bystander activated. Nevertheless, the method is expected to be a valuable tool to evaluate and quantify vaccine responses to current and new chicken vaccines in the future.

A high-mobility, low-cost phenotype defines human effector-memory CD8+ T cells.

T cells move randomly ("random-walk"), a characteristic thought to be integral to their function. Using migration assays and time-lapse microscopy, we found that CD8+ T cells lacking the lymph node homing receptors CCR7 and CD62L migrate more efficiently in transwell assays, and that these same cells are characterized by a high frequency of cells exhibiting random crawling activity under culture conditions mimicking the interstitial/extravascular milieu, but not when examined on endothelial cells. To assess the energy efficiency of cells crawling at a high frequency, we measured mRNA expression of genes key to mitochondrial energy metabolism (peroxisome proliferator-activated receptor gamma coactivator 1beta [PGC-1beta], estrogen-related receptor alpha [ERRalpha], cytochrome C, ATP synthase, and the uncoupling proteins [UCPs] UCP-2 and -3), quantified ATP contents, and performed calorimetric analyses. Together these assays indicated a high energy efficiency of the high crawling frequency CD8+ T-cell population, and identified differentially regulated heat production among nonlymphoid versus lymphoid homing CD8+ T cells.

Flow cytometric lymphocyte subset enumeration: 10 years of external quality assessment in the Benelux countries.

A biannual external quality assessment (EQA) scheme for flow cytometric lymphocyte immunophenotyping is operational in the Benelux countries since 1996. We studied the effects of the methods used on assay outcome, and whether or not this EQA exercise was effective in reducing between-laboratory variation. Eighty test samples were distributed in 20 biannual send-outs. Per send-out, 50-71 participants were requested to enumerate CD3+, CD4+, and CD8+ T cells, B cells, and NK cells, and to provide methodological details. Participants received written debriefings with personalized recommendations after each send-out. For this report, data were analyzed using robust multivariate regression. Five variables were associated with significant positive or negative bias of absolute lymphocyte subset counts: (i) platform methodology (i.e., single-platform assays yielded lower CD4+ and CD8+ T-cell counts than did dual-platform assays); (ii) sample preparation technique (i.e., assays based on mononuclear cells isolation yielded lower T-cell counts than those based on red cell lysis); (iii) gating strategies based on CD45 and sideward scatter gating of lymphocytes yielded higher CD4+ T-cell counts than those based on "backgating" of lymphocytes guided by CD45 and CD14); (iv) stabilized samples were generally associated with higher lymphocyte subset counts than nonstabilized samples; and (v) laboratory. Platform methodology, sample stabilization, and laboratory also affected assay variability. With time, assay variability tended to decline; this trend was significant for B-cell counts only. In addition, significant bias and variability of results, independent of the variables tested for in this analysis, were also associated with individual laboratories. In spite of our recommendations, participants tended to standardize their techniques mainly with respect to sample preparation and gating strategies, but less with absolute counting techniques. Failure to fully standardize protocols may have led to only modest reductions in variability of results between laboratories.

A multicenter evaluation of the PanLeucogating method and the use of generic monoclonal antibody reagents for CD4 enumeration in HIV-infected patients in Thailand.

BACKGROUND: The current method of CD4 enumeration in Thailand, based on the three-tube, three-color method recommended by the Centers for Disease Control and Prevention, is expensive and thus unavailable to most patients who have the human immunodeficiency virus (HIV). Less expensive, simpler protocols (i.e., PanLeucogating and primary CD4 gating) have been described but require more published validation data to gain widespread acceptance. We describe a multicenter evaluation of the PanLeucogating method. METHODS: The PanLeucogating method using generic reagents was evaluated in comparison with the standard three-tube, three-color method using commercial reagents. Percentage of CD4+ T cells among lymphocytes and absolute CD4+ T-cell counts were determined in 611 HIV-infected individuals recruited from four sites. Linear regression and Bland-Altman tests were used for statistical analysis. RESULTS: The correlation of percentage of CD4+ T cells and absolute CD4+ T-cell counts obtained with the PanLeucogating strategy and the standard predicate method was high (r2 = 0.96 and 0.95, respectively, for the entire study population and r2 > 0.95 and 0.93, respectively, for each study group). Absolute CD4+ T-cell counts of the overall study pool and of the two subdivisions of absolute CD4+ T-cell counts (i.e., 0-250 cells/microl and > 250 cells/microl) derived from the two methods demonstrated excellent agreement, with mean biases of +18 cells/microl, +11 cells/microl, and +24 cells/microl, respectively. CONCLUSIONS: These observations demonstrate that CD4 enumeration by PanLeucogating is reliable and can be performed to an identical standard in a quality-assured network of collaborating laboratories as a new cost-effective approach to HIV monitoring.

Deceptive multilineage reconstitution analysis of mice transplanted with hemopoietic stem cells, and implications for assessment of stem cell numbers and lineage potentials.

Hemopoietic stem cells (HSC) are identified through their unique ability, at the single cell level, to long-term reconstitute all blood cell lineages. Sustained myeloid reconstitution is considered the hallmark of HSC, because myeloid progenitors and their progeny have very short half-lives. Here we demonstrate that the established practice of relying on RB6-8C5 as a myeloid specific Ab can result in overestimation of HSC frequencies because the RB6-8C5 Ab also detects Ags expressed on a sizeable population of CD3(+)CD8(+) T cells, constitutively as well as following transplantation. Likewise, a high fraction of mice transplanted with limiting numbers of ex vivo expanded Lin(-)Sca(+)kit(+)CD34(-) HSC show long-term RB6-8C5(+)CD3(+) (lymphoid) but no RB6-8C5(+)CD3(-) (myeloid) reconstitution. Most noteworthy, the use of RB6-8C5 as a myeloid specific Ab can be deceptive by implicating the existence of lineage-restricted HSC capable of long-term reconstituting the myeloid and T, but not B, cell lineage. Because cross-lineage expression of "lineage-specific" markers is unlikely to be unique to the blood system, claims of unexpected cell fates should be substantiated not only by acquisition of lineage-specific markers, but also absence of markers of other lineages normally derived from the investigated stem cells.