Quantifying in situ adaptive immune cell cognate interactions in humans.

Two-photon excitation microscopy (TPEM) has revolutionized the understanding of adaptive immunity. However, TPEM usually requires animal models and is not amenable to the study of human disease. The recognition of antigen by T cells requires cell contact and is associated with changes in T cell shape. We postulated that by capturing these features in fixed tissue samples, we could quantify in situ adaptive immunity. Therefore, we used a deep convolutional neural network to identify fundamental distance and cell-shape features associated with cognate help (cell-distance mapping (CDM)). In mice, CDM was comparable to TPEM in discriminating cognate T cell-dendritic cell (DC) interactions from non-cognate T cell-DC interactions. In human lupus nephritis, CDM confirmed that myeloid DCs present antigen to CD4+ T cells and identified plasmacytoid DCs as an important antigen-presenting cell. These data reveal a new approach with which to study human in situ adaptive immunity broadly applicable to autoimmunity, infection, and cancer.

Transendothelial migration of lymphocytes mediated by intraendothelial vesicle stores rather than by extracellular chemokine depots.

Chemokines presented by the endothelium are critical for integrin-dependent adhesion and transendothelial migration of naive and memory lymphocytes. Here we found that effector lymphocytes of the type 1 helper T cell (T(H)1 cell) and type 1 cytotoxic T cell (T(C)1 cell) subtypes expressed adhesive integrins that bypassed chemokine signals and established firm arrests on variably inflamed endothelial barriers. Nevertheless, the transendothelial migration of these lymphocytes strictly depended on signals from guanine nucleotide-binding proteins of the G(i) type and was promoted by multiple endothelium-derived inflammatory chemokines, even without outer endothelial surface exposure. Instead, transendothelial migration-promoting endothelial chemokines were stored in vesicles docked on actin fibers beneath the plasma membranes and were locally released within tight lymphocyte-endothelial synapses. Thus, effector T lymphocytes can cross inflamed barriers through contact-guided consumption of intraendothelial chemokines without surface-deposited chemokines or extraendothelial chemokine gradients.

The Immune Adaptor SLP-76 Binds to SUMO-RANGAP1 at Nuclear Pore Complex Filaments to Regulate Nuclear Import of Transcription Factors in T Cells.

While immune cell adaptors regulate proximal T cell signaling, direct regulation of the nuclear pore complex (NPC) has not been reported. NPC has cytoplasmic filaments composed of RanGAP1 and RanBP2 with the potential to interact with cytoplasmic mediators. Here, we show that the immune cell adaptor SLP-76 binds directly to SUMO-RanGAP1 of cytoplasmic fibrils of the NPC, and that this interaction is needed for optimal NFATc1 and NF-κB p65 nuclear entry in T cells. Transmission electron microscopy showed anti-SLP-76 cytoplasmic labeling of the majority of NPCs in anti-CD3 activated T cells. Further, SUMO-RanGAP1 bound to the N-terminal lysine 56 of SLP-76 where the interaction was needed for optimal RanGAP1-NPC localization and GAP exchange activity. While the SLP-76-RanGAP1 (K56E) mutant had no effect on proximal signaling, it impaired NF-ATc1 and p65/RelA nuclear entry and in vivo responses to OVA peptide. Overall, we have identified SLP-76 as a direct regulator of nuclear pore function in T cells.

A general chemical crosslinking strategy for structural analyses of weakly interacting proteins applied to preTCR-pMHC complexes.

T lymphocytes discriminate between healthy and infected or cancerous cells via T-cell receptor-mediated recognition of peptides bound and presented by cell-surface-expressed major histocompatibility complex molecules (MHCs). Pre-T-cell receptors (preTCRs) on thymocytes foster development of αßT lymphocytes through their ß chain interaction with MHC displaying self-peptides on thymic epithelia. The specific binding of a preTCR with a peptide-MHC complex (pMHC) has been identified previously as forming a weak affinity complex with a distinct interface from that of mature αßTCR. However, a lack of appropriate tools has limited prior efforts to investigate this unique interface. Here we designed a small-scale linkage screening protocol using bismaleimide linkers for determining residue-specific distance constraints between transiently interacting protein pairs in solution. Employing linkage distance restraint-guided molecular modeling, we report the oriented solution docking geometry of a preTCRß-pMHC interaction. The linkage model of preTCRß-pMHC complex was independently verified with paramagnetic pseudocontact chemical shift (PCS) NMR of the unlinked protein mixtures. Using linkage screens, we show that the preTCR binds with differing affinities to peptides presented by MHC in solution. Moreover, the C-terminal peptide segment is a key determinant in preTCR-pMHC recognition. We also describe the process for future large-scale production and purification of the linked constructs for NMR, X-ray crystallography, and single-molecule electron microscopy studies.

Augmented T-cell mitochondrial reactive oxygen species in adults with major depressive disorder.

The prevalence of major depressive disorder (MDD) is highest in young adulthood, an effect that has been magnified by the COVID-19 pandemic. Importantly, individuals with MDD are at a greater risk of developing cardiovascular disease (CVD). Accumulating evidence supports immune system dysregulation as a major contributor to the elevated CVD risk in older adults with MDD; however, whether this is present in young adults with MDD without comorbid disease remains unclear. Interestingly, recent data suggest augmented T-cell mitochondrial reactive oxygen species (T-cell mitoROS) as a potent driver of immune dysregulation in animal models of psychiatric disease. With this background in mind, we tested the hypothesis that young adults with MDD would have augmented T-cell mitoROS and circulating proinflammatory cytokines compared with healthy young adults without MDD (HA). Whole blood was drawn from 14 young adults with MDD (age: 23 ± 2 yr) and 11 HA (age: 22 ± 1 yr). T-cell mitoROS (MitoSOX red; total: CD3+, T-helper: CD4+, T cytotoxic: CD8+) and serum cytokines were assessed by flow cytometry. Total T-cell mitoROS was significantly greater in adults with MDD compared with HA [median: 14,089 arbitrary units (AU); median: 1,362 AU, P = 0.01]. Likewise, both T-helper and T-cytotoxic cell mitoROS were significantly greater in adults with MDD compared with HA (both: P < 0.05). There were no differences in circulating cytokines between groups (all cytokines: P > 0.05). Collectively, these findings suggest that elevated T-cell mitoROS may represent an early marker of immune system dysregulation in young, otherwise healthy, adults with MDD.NEW & NOTEWORTHY To our knowledge, we provide the first evidence of augmented T-cell mitochondrial reactive oxygen species (T-cell mitoROS) in young, otherwise healthy adults with MDD. Although the elevated T-cell mitoROS did not correspond to a proinflammatory profile, these findings suggest that elevated T-cell mitoROS may be an early marker of immune system dysregulation in young adults with MDD.

TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation.

The organization and dynamics of receptors and other molecules in the plasma membrane are not well understood. Here we analyzed the spatio-temporal dynamics of T cell antigen receptor (TCR) complexes and linker for activation of T cells (Lat), a key adaptor molecule in the TCR signaling pathway, in T cell membranes using high-speed photoactivated localization microscopy, dual-color fluorescence cross-correlation spectroscopy and transmission electron microscopy. In quiescent T cells, both molecules existed in separate membrane domains (protein islands), and these domains concatenated after T cell activation. These concatemers were identical to signaling microclusters, a prominent hallmark of T cell activation. This separation versus physical juxtapositioning of receptor domains and domains containing downstream signaling molecules in quiescent versus activated T cells may be a general feature of plasma membrane-associated signal transduction.

4D electron microscopy of T cell activation.

T cells can be controllably stimulated through antigen-specific or nonspecific protocols. Accompanying functional hallmarks of T cell activation can include cytoskeletal reorganization, cell size increase, and cytokine secretion. Photon-induced near-field electron microscopy (PINEM) is used to image and quantify evanescent electric fields at the surface of T cells as a function of various stimulation conditions. While PINEM signal strength scales with multiple of the biophysical changes associated with T cell functional activation, it mostly strongly correlates with antigen-engagement of the T cell receptors, even under conditions that do not lead to functional T cell activation. PINEM image analysis suggests that a stimulation-induced reorganization of T cell surface structure, especially over length scales of a few hundred nanometers, is the dominant contributor to these PINEM signal changes. These experiments reveal that PINEM can provide a sensitive label-free probe of nanoscale cellular surface structures.

How does T cell receptor clustering impact on signal transduction?

The essential function of the T cell receptor (TCR) is to translate the engagement of peptides on the major histocompatibility complex (pMHC) into appropriate intracellular signals through the associated cluster of differentiation 3 (CD3) complex. The spatial organization of the TCR-CD3 complex in the membrane is thought to be a key regulatory element of signal transduction, raising the question of how receptor clustering impacts on TCR triggering. How signal transduction at the TCR-CD3 complex encodes the quality and quantity of pMHC molecules is not fully understood. This question can be approached by reconstituting T cell signaling in model and cell membranes and addressed by single-molecule imaging of endogenous proteins in T cells. We highlight such methods and further discuss how TCR clustering could affect pMHC rebinding rates, the local balance between kinase and phosphatase activity and/or the lipid environment to regulate the signal efficiency of the TCR-CD3 complex. We also examine whether clustering could affect the conformation of cytoplasmic CD3 tails through a biophysical mechanism. Taken together, we highlight how the spatial organization of the TCR-CD3 complex - addressed by reconstitution approaches - has emerged as a key regulatory element in signal transduction of this archetypal immune receptor.

Genome-wide association study of individual differences of human lymphocyte profiles using large-scale cytometry data.

Human immune systems are very complex, and the basis for individual differences in immune phenotypes is largely unclear. One reason is that the phenotype of the immune system is so complex that it is very difficult to describe its features and quantify differences between samples. To identify the genetic factors that cause individual differences in whole lymphocyte profiles and their changes after vaccination without having to rely on biological assumptions, we performed a genome-wide association study (GWAS), using cytometry data. Here, we applied computational analysis to the cytometry data of 301 people before receiving an influenza vaccine, and 1, 7, and 90 days after the vaccination to extract the feature statistics of the lymphocyte profiles in a nonparametric and data-driven manner. We analyzed two types of cytometry data: measurements of six markers for B cell classification and seven markers for T cell classification. The coordinate values calculated by this method can be treated as feature statistics of the lymphocyte profile. Next, we examined the genetic basis of individual differences in human immune phenotypes with a GWAS for the feature statistics, and we newly identified seven significant and 36 suggestive single-nucleotide polymorphisms associated with the individual differences in lymphocyte profiles and their change after vaccination. This study provides a new workflow for performing combined analyses of cytometry data and other types of genomics data.

Novel insight from the first lung transplant of a COVID-19 patient.

BACKGROUND: To reveal detailed histopathological changes, virus distributions, immunologic properties and multi-omic features caused by SARS-CoV-2 in the explanted lungs from the world's first successful lung transplantation of a COVID-19 patient. MATERIALS AND METHODS: A total of 36 samples were collected from the lungs. Histopathological features and virus distribution were observed by optical microscope and transmission electron microscope (TEM). Immune cells were detected by flow cytometry and immunohistochemistry. Transcriptome and proteome approaches were used to investigate main biological processes involved in COVID-19-associated pulmonary fibrosis. RESULTS: The histopathological changes of the lung tissues were characterized by extensive pulmonary interstitial fibrosis and haemorrhage. Viral particles were observed in the cytoplasm of macrophages. CD3+ CD4- T cells, neutrophils, NK cells, γ/δ T cells and monocytes, but not B cells, were abundant in the lungs. Higher levels of proinflammatory cytokines iNOS, IL-1ß and IL-6 were in the area of mild fibrosis. Multi-omics analyses revealed a total of 126 out of 20,356 significant different transcription and 114 out of 8,493 protein expression in lung samples with mild and severe fibrosis, most of which were related to fibrosis and inflammation. CONCLUSIONS: Our results provide novel insight that the significant neutrophil/ CD3+ CD4- T cell/ macrophage activation leads to cytokine storm and severe fibrosis in the lungs of COVID-19 patient and may contribute to a better understanding of COVID-19 pathogenesis.

Role and Function of T Cell-Derived Exosomes and Their Therapeutic Value.

Exosomes are membrane-bound extracellular vesicles that are produced in the endosomal compartment of most eukaryotic cells. Containing proteins, RNA, and DNA, exosomes mediate intercellular communication between different cell types by transferring their contents and thus are involved in numerous physiological and pathological processes. T cells are an indispensable part of adaptive immunity, and the functions of T cell-derived exosomes have been widely studied. In the more than three decades since the discovery of exosomes, several studies have revealed that T cell-derived exosomes play a novel role in cell-to-cell signaling, especially in inflammatory responses, autoimmunity, and infectious diseases. In this review, we will summarize the function of T cell-derived exosomes and their therapeutic potential.

Membrane lipid order of sub-synaptic T cell vesicles correlates with their dynamics and function.

During an immune response, T cells survey antigen presenting cells for antigenic peptides via the formation of an interface known as an immunological synapse. Among the complex and dynamic biophysical phenomena occurring at this interface is the trafficking of sub-synaptic vesicles carrying a variety of proximal signalling molecules. Here, we show that rather than being a homogeneous population, these vesicles display a diversity of membrane lipid order profiles, as measured using the environmentally sensitive dye di-4-ANEPPDHQ and multi-spectral TIRF microscopy. Using live-cell imaging, vesicle tracking and a variety of small molecule drugs to manipulate components of the actin and tubulin cytoskeleton, we show that the membrane lipid order of these vesicles correlate with their dynamics. Furthermore, we show that the key proximal signalling molecule Linker for Activation of T cells (LAT) is enriched in specific vesicle populations as defined by their higher membrane order. These results imply that vesicle lipid order may represent a novel regulatory mechanism for the sorting and trafficking of signalling molecules at the immunological synapse, and, potentially, other cellular structures.

A Reproducible, Objective Method Using MitoTracker® Fluorescent Dyes to Assess Mitochondrial Mass in T Cells by Flow Cytometry.

MitoTracker ® dyes are fluorescent compounds that allow cellular mitochondrial content to be measured semi-quantitatively by flow cytometry and have been used extensively in immunology publications. However, the parameters commonly reported, mean or median fluorescence intensity and percentage of cells that are MitoTracker® "high", can be influenced by variability in cytometer setup, dye stability, and operator subjectivity, making it difficult to compare data between experiments. Here, we describe a method to identify MitoTracker® "high" populations in an objective manner. When analyzing data, we first removed outliers using a pre-specified threshold, determined the fluorescence intensity of the brightest and dimmest events to obtain the fluorescence range and then gated cells within the top 90% of this range. This strategy substantially reduced variability between technical replicates and produced consistent results when data were analyzed by different operators. Consistent with previous reports and other analysis strategies, this analysis method demonstrated that within an individual, CD4+ T cells exhibit significantly higher mitochondrial mass than CD8+ T cells. Objective gating increases the reliability and utility of data generated using MitoTracker® dyes. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Clickable Methyltetrazine-Indocarbocyanine Lipids: A Multicolor Tool Kit for Efficient Modifications of Cell Membranes.

Cell-based therapeutics are one of the most promising and exciting breakthroughs in modern medicine. Modification of the cell surface with ligands, biologics, drugs, and nanoparticles can further enhance the functionality. Previously, we described the synthesis of a dioctadecyl indocarbocyanine Cy3 analog (aminomethyl-DiI) for efficient and stable modification (painting) of mouse erythrocytes with small molecules, enzymes, and biologics. Here, we synthesized a near-infrared aminomethyl dioctadecyl derivative of Cy7 (aminomethyl-DOCy7) and systematically compared it to aminomethyl-DiI as an anchor for the modification of human erythrocytes, Jurkat cells, and primary T cells with immunoglobulin G. To enable copper-free click chemistry modification of cell membranes, we conjugated a methyltetrazine (MTz) group to the amino-indocyanine lipids via a polyethylene glycol (PEG) linker. DOCy7-PEG3400-MTz showed over 99% modification efficiency of human red blood cells (RBCs) at 25 µM. Reaction of trans-cyclooctene (TCO) modified immunoglobulin G (IgG) with DOCy7-PEG4-MTz-modified RBCs (2-step method) resulted in ∼80,000 IgG molecules per erythrocyte, whereas modification with a preconjugated DOCy7-PEG3400-IgG construct (1-step method) resulted in ∼20,000 IgG molecules per erythrocyte as detected by immuno dot-blot. The number of IgG/RBC was controlled by the concentration of IgG. The incubation of RBCs with DiI-PEG3400-MTz resulted in a similar number of IgG/RBC. Modification of the T-lymphocyte cell line Jurkat with IgG resulted in ∼1 × 106 IgG/cell with the 1-step and 2-step methods, and the efficiency was similar for DOCy7 and DiI constructs. Finally, we used DOCy7 and DiI constructs to demonstrate efficient modification of primary CD3+T cells from healthy donors. In conclusion, click indocarbocyanine conjugates represent a novel multicolor chemical biology tool kit for efficient surface modification of different cells types and can be used for potential imaging and drug delivery applications involving engineered cells.

Junctophilin-4, a component of the endoplasmic reticulum-plasma membrane junctions, regulates Ca2+ dynamics in T cells.

Orai1 and stromal interaction molecule 1 (STIM1) mediate store-operated Ca(2+) entry (SOCE) in immune cells. STIM1, an endoplasmic reticulum (ER) Ca(2+) sensor, detects store depletion and interacts with plasma membrane (PM)-resident Orai1 channels at the ER-PM junctions. However, the molecular composition of these junctions in T cells remains poorly understood. Here, we show that junctophilin-4 (JP4), a member of junctional proteins in excitable cells, is expressed in T cells and localized at the ER-PM junctions to regulate Ca(2+) signaling. Silencing or genetic manipulation of JP4 decreased ER Ca(2+) content and SOCE in T cells, impaired activation of the nuclear factor of activated T cells (NFAT) and extracellular signaling-related kinase (ERK) signaling pathways, and diminished expression of activation markers and cytokines. Mechanistically, JP4 directly interacted with STIM1 via its cytoplasmic domain and facilitated its recruitment into the junctions. Accordingly, expression of this cytoplasmic fragment of JP4 inhibited SOCE. Furthermore, JP4 also formed a complex with junctate, a Ca(2+)-sensing ER-resident protein, previously shown to mediate STIM1 recruitment into the junctions. We propose that the junctate-JP4 complex located at the junctions cooperatively interacts with STIM1 to maintain ER Ca(2+) homeostasis and mediate SOCE in T cells.

Three-dimensional localization of T-cell receptors in relation to microvilli using a combination of superresolution microscopies.

Leukocyte microvilli are flexible projections enriched with adhesion molecules. The role of these cellular projections in the ability of T cells to probe antigen-presenting cells has been elusive. In this study, we probe the spatial relation of microvilli and T-cell receptors (TCRs), the major molecules responsible for antigen recognition on the T-cell membrane. To this end, an effective and robust methodology for mapping membrane protein distribution in relation to the 3D surface structure of cells is introduced, based on two complementary superresolution microscopies. Strikingly, TCRs are found to be highly localized on microvilli, in both peripheral blood human T cells and differentiated effector T cells, and are barely found on the cell body. This is a decisive demonstration that different types of T cells universally localize their TCRs to microvilli, immediately pointing to these surface projections as effective sensors for antigenic moieties. This finding also suggests how previously reported membrane clusters might form, with microvilli serving as anchors for specific T-cell surface molecules.

Qualitative and quantitative analysis of PMN/T-cell interactions by InFlow and super-resolution microscopy.

Neutrophils or polymorphonuclear cells (PMN) eliminate bacteria via phagocytosis and/or NETosis. Apart from these conventional roles, PMN also have immune-regulatory functions. They can transdifferentiate and upregulate MHCII as well as ligands for costimulatory receptors which enables them to behave as antigen presenting cells (APC). The initial step for activating T-cells is the formation of an immune synapse between T-cells and antigen-presenting cells. However, the immune synapse that develops at the PMN/T-cell contact zone is as yet hardly investigated due to the non-availability of methods for analysis of large number of PMN interactions. In order to overcome these obstacles, we introduce here a workflow to analyse the immune synapse of primary human PMN and T-cells using multispectral imaging flow cytometry (InFlow microscopy) and super-resolution microscopy. For that purpose, we used CD3 and CD66b as the lineage markers for T-cells and PMN, respectively. Thereafter, we applied and critically discussed various "masks" for identification of T-cell PMN interactions. Using this approach, we found that a small fraction of transdifferentiated PMN (CD66b+CD86high) formed stable PMN/T-cell conjugates. Interestingly, while both CD3 and CD66b accumulation in the immune synapse was dependent on the maturation state of the PMN, only CD3 accumulation was greatly enhanced by the presence of superantigen. The actin cytoskeleton was weakly rearranged at the PMN side on the immune synapse upon contact with a T-cell in the presence of superantigen. A more detailed analysis using super-resolution microscopy (structured-illumination microscopy, SIM) confirmed this finding. Together, we present an InFlow microscopy based approach for the large scale analysis of PMN/T-cell interactions and - combined with SIM - a possibility for an in-depth analysis of protein translocation at the site of interactions.

Maternal diet during pregnancy and micronuclei frequency in peripheral blood T lymphocytes in mothers and newborns (Rhea cohort, Crete).

PURPOSE: The study assessed whether diet and adherence to cancer prevention guidelines during pregnancy were associated with micronucleus (MN) frequency in mothers and newborns. MN is biomarkers of early genetic effects that have been associated with cancer risk in adults. METHODS: A total of 188 mothers and 200 newborns from the Rhea cohort (Greece) were included in the study. At early-mid pregnancy, we conducted personal interviews and a validated food frequency questionnaire was completed. With this information, we constructed a score reflecting adherence to the World Cancer Research Fund/American Institute for Cancer Research cancer prevention guidelines on diet, physical activity and body fatness. At delivery, maternal and/or cord blood was collected to measure DNA and hemoglobin adducts of dietary origin and frequencies of MN in binucleated and mononucleated T lymphocytes (MNBN and MNMONO). RESULTS: In mothers, higher levels of red meat consumption were associated with increased MNBN frequency [2nd tertile IRR = 1.34 (1.00, 1.80), 3rd tertile IRR = 1.33 (0.96, 1.85)] and MNMONO frequency [2nd tertile IRR = 1.53 (0.84, 2.77), 3rd tertile IRR = 2.69 (1.44, 5.05)]. The opposite trend was observed for MNBN in newborns [2nd tertile IRR = 0.64 (0.44, 0.94), 3rd tertile IRR = 0.68 (0.46, 1.01)], and no association was observed with MNMONO. Increased MN frequency in pregnant women with high red meat consumption is consistent with previous knowledge. CONCLUSIONS: Our results also suggest exposure to genotoxics during pregnancy might affect differently mothers and newborns. The predictive value of MN as biomarker for childhood cancer, rather than adulthood, remains unclear. With few exceptions, the association between maternal carcinogenic exposures during pregnancy and childhood cancer or early biologic effect biomarkers remains poorly understood.

Optimization of mass cytometry sample cryopreservation after staining.

The advent of mass cytometry has facilitated highly multi-parametric single-cell analysis allowing for the deep assessment of cellular diversity. While the data and analytical power of this approach are well described, associated technical and experimental hurdles remain. Issues like equipment breakdown and sampling of large-scale batches, which may require multiple days of data acquisition, are minor but critical obstacles that prompt a technical solution, especially when dealing with precious samples. An ability to cryopreserve mass cytometry samples that have already been stained would alleviate numerous technical limitations we face with currently used sample-handling approaches. Here, we evaluated two protocols for freezing of already-stained and fixed cellular samples and compared them with standard sample refrigeration in staining buffer. A comprehensive human T cell staining phenotypic and functional profiling panel was used and the signal intensity and reliability of each marker was assessed over a 4-week period. In general, cellular viability, DNA Ir-Intercalator and barcode staining were minimally affected by freezing compared to refrigeration, and the signal intensities for cell surface markers and receptors were not compromised. Intracellular cytokine staining did show some decreases in signal intensity after freezing, with the decreases more prominent in a methanol-based protocol compared to a protocol involving the use of 10% DMSO in FBS. We conclude that freezing already-stained samples suspended in 10% DMSO in FBS is practical and efficient way to preserve already-stained samples when needed. © 2016 International Society for Advancement of Cytometry.

Computer-assisted quantification of CD3+ T cells in follicular lymphoma.

The advance of high resolution digital scans of pathology slides allowed development of computer based image analysis algorithms that may help pathologists in IHC stains quantification. While very promising, these methods require further refinement before they are implemented in routine clinical setting. Particularly critical is to evaluate algorithm performance in a setting similar to current clinical practice. In this article, we present a pilot study that evaluates the use of a computerized cell quantification method in the clinical estimation of CD3 positive (CD3+) T cells in follicular lymphoma (FL). Our goal is to demonstrate the degree to which computerized quantification is comparable to the practice of estimation by a panel of expert pathologists. The computerized quantification method uses entropy based histogram thresholding to separate brown (CD3+) and blue (CD3-) regions after a color space transformation. A panel of four board-certified hematopathologists evaluated a database of 20 FL images using two different reading methods: visual estimation and manual marking of each CD3+ cell in the images. These image data and the readings provided a reference standard and the range of variability among readers. Sensitivity and specificity measures of the computer's segmentation of CD3+ and CD- T cell are recorded. For all four pathologists, mean sensitivity and specificity measures are 90.97 and 88.38%, respectively. The computerized quantification method agrees more with the manual cell marking as compared to the visual estimations. Statistical comparison between the computerized quantification method and the pathologist readings demonstrated good agreement with correlation coefficient values of 0.81 and 0.96 in terms of Lin's concordance correlation and Spearman's correlation coefficient, respectively. These values are higher than most of those calculated among the pathologists. In the future, the computerized quantification method may be used to investigate the relationship between the overall architectural pattern (i.e., interfollicular vs. follicular) and outcome measures (e.g., overall survival, and time to treatment). © 2017 International Society for Advancement of Cytometry.