A Discrete Chain Graph Model for 3d+t Cell Tracking with High Misdetection Robustness

AbstractIn the past decades, immunotherapies against cancers made impressive progress. Immunotherapy includes a broad range of interventions that can be separated into two major groups: cell-based immunotherapies, such as adoptive T cell therapies and stem cell therapies, and immunomodulatory molecular therapies such as checkpoint inhibitors and cytokine therapies. Genetic engineering techniques that transduce T cells with a cancer-antigen-specific T cell receptor or chimeric antigen receptor have expanded to other cell types, and further modulation of the cells to enhance cancer targeting properties has been explored. Because cell-based immunotherapies rely on cells migrating to target organs or tissues, there is a growing interest in imaging technologies that non-invasively monitor transferred cells in vivo. Here, we review whole-body imaging methods to assess cell-based immunotherapy using a variety of examples. Following a review of preclinically used cell tracking technologies, we consider the status of their clinical translation.

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Non-invasive cell-tracking methods for adoptive T cell therapies

Drug Discovery Today ◽

10.1016/j.drudis.2021.10.012 ◽

2021 ◽

Author(s):

Jelter Van Hoeck ◽

Christian Vanhove ◽

Stefaan C. De Smedt ◽

Koen Raemdonck

Keyword(s):

T Cell ◽

Cell Tracking ◽

Cell Therapies ◽

Non Invasive ◽

Invasive Cell

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T-cell calcium dynamics visualized in a ratiometric tdTomato-GCaMP6f transgenic reporter mouse

eLife ◽

10.7554/elife.32417 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 18

Author(s):

Tobias X Dong ◽

Shivashankar Othy ◽

Amit Jairaman ◽

Jonathan Skupsky ◽

Angel Zavala ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Tracking ◽

Cell Types ◽

Cell Receptor ◽

Receptor Activation ◽

Specific Cell ◽

Transgenic Expression ◽

Reporter Mouse

Calcium is an essential cellular messenger that regulates numerous functions in living organisms. Here, we describe development and characterization of ‘Salsa6f’, a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic Ca2+, and a transgenic Ca2+ reporter mouse with Salsa6f targeted to the Rosa26 locus for Cre-dependent expression in specific cell types. The development and function of T cells was unaffected in Cd4-Salsa6f mice. We describe Ca2+ signals reported by Salsa6f during T cell receptor activation in naive T cells, helper Th17 T cells and regulatory T cells, and Ca2+ signals mediated in T cells by an activator of mechanosensitive Piezo1 channels. Transgenic expression of Salsa6f enables ratiometric imaging of Ca2+ signals in complex tissue environments found in vivo. Two-photon imaging of migrating T cells in the steady-state lymph node revealed both cell-wide and localized sub-cellular Ca2+ transients (‘sparkles’) as cells migrate.

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T Cell Calcium Dynamics Visualized in a Ratiometric tdTomato-GCaMP6f Transgenic Reporter Mouse

10.1101/203950 ◽

2017 ◽

Author(s):

Tobias X. Dong ◽

Shivashankar Othy ◽

Amit Jairaman ◽

Jonathan Skupsky ◽

Angel Zavala ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Tracking ◽

Cell Types ◽

Cell Receptor ◽

Functional Expression ◽

Specific Cell ◽

Transgenic Expression ◽

Reporter Mouse

AbstractCalcium is an essential cellular messenger that regulates numerous functions in living organisms. Here we describe development and characterization of “Salsa6f”, a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic Ca2+, and a transgenic Ca2+ reporter mouse with Salsa6f floxed and targeted to the Rosa26 locus for expression in specific cell types. Using CD4-Cre-Salsa6f mice, we report normal development and function of T cells expressing Salsa6f and demonstrate Ca2+ signaling dynamics during T cell receptor engagement in naïve T cells, helper Th17 T cells and regulatory T cells. Salsa6f expression also revealed functional expression of mechanosensitive Piezo1 channels in T cells. Transgenic expression of Salsa6f enables ratiometric imaging of Ca2+ signals in complex tissue environments found in vivo. Deep tissue two-photon imaging of T cells in the steady-state lymph node revealed a highly localized Ca2+ signaling behavior (“sparkles”) as cells migrate.

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The fate of self-reactive B cells depends primarily on the degree of antigen receptor engagement and availability of T cell help.

Journal of Experimental Medicine ◽

10.1084/jem.183.5.2313 ◽

1996 ◽

Vol 183 (5) ◽

pp. 2313-2328 ◽

Cited By ~ 189

Author(s):

D A Fulcher ◽

A B Lyons ◽

S L Korn ◽

M C Cook ◽

C Koleda ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

Life Span ◽

Cell Tracking ◽

Soluble Form ◽

Critical Threshold ◽

Short Life ◽

Short Life Span ◽

T Cell Help ◽

Self Antigen

Self-reactive B cells from tolerant double-transgenic (Dbl-Tg) mice coexpressing hen egg lysozyme (HEL) and rearranged anti-HEL immunoglobulin genes have a relatively short life span when compared to normal B cells, irrespective of whether they are exposed to antigen in multivalent membrane-bound form (mHEL-Dbl-Tg mice) or soluble form (sHEL-Dbl-Tg mice). The factors responsible for determining the fate of these B cells after encounter with self-antigen were investigated using a cell-tracking technique in which anti-HEL Ig-Tg spleen cells were labeled with the intracellular dye 5-carboxyfluorescein diacetate-succinimidyl ester (CFSE) and injected either into non-Tg recipients or a variety of HEL-Tg hosts. In non-Tg recipients, HEL-binding B cells persisted in the circulation and could be detected in the follicles of the spleen for at least 5 d. On transfer into either mHEL-Tg or sHEL-Tg hosts, they underwent activation and then rapidly disappeared from the blood and spleen over the next 3 d, consistent with the short life span reported previously. Immunohistology of spleens from sHEL-Tg recipients indicated that the transferred B cells had migrated to the outer margins of the periarteriolar lymphoid sheath (PALS), where they were detectable for 24 h before being lost. The positioning of B cells in the outer PALS depended on a critical threshold of Ig receptor binding corresponding to a serum HEL concentration between 0.5 and 15 ng/ml, but was not restricted to endogenously expressed HEL in that the same migratory pattern was observed after transfer into non-Tg recipients given exogenous (foreign) HEL. Moreover, bone marrow-derived immature Ig-Tg B cells homed to the outer PALS of sHEL-Tg mice and then disappeared at the same rate as mature B cells, indicating that the stage of maturation did not influence the fate of self-reactive B cells in a tolerant environment. On the other hand, HEL-binding B cells transferred into sHEL-Dbl-Tg recipients persisted over the 3-d period of study, apparently due to insufficient availability of antigen, as indicated by the fact that the degree of Ig receptor downregulation on the transferred B cells was much less than in sHEL-Tg recipients. If T cell help was provided to Ig-Tg B cells at the time of transfer into sHEL-Tg recipients in the form of preactivated CD4+ T cells specific for major histocompatibility complex-peptide complexes on the B cell surface, HEL-binding B cells migrated through the outer PALS of the spleen to the follicle, where they formed germinal centers, or to adjacent red pulp, where they formed proliferative foci and secreted significant amounts of anti-HEL antibody. Taken together, these results indicated that the outcome of the interaction between self-antigen and B cells is largely determined by a combination of the degree of receptor engagement and availability of T cell help.

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CAR T-cells that target acute B-lineage leukemia irrespective of CD19 expression

Leukemia ◽

10.1038/s41375-020-0792-2 ◽

2020 ◽

Vol 35 (1) ◽

pp. 75-89 ◽

Cited By ~ 10

Author(s):

Kristen Fousek ◽

Junji Watanabe ◽

Sujith K. Joseph ◽

Ann George ◽

Xingyue An ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Tracking ◽

Lymphoblastic Leukemia ◽

Target Cells ◽

T Cell Therapy ◽

Car T Cells ◽

Car T ◽

B Lineage ◽

Cd19 Expression

AbstractChimeric antigen receptor (CAR) T-cells targeting CD19 demonstrate remarkable efficacy in treating B-lineage acute lymphoblastic leukemia (BL-ALL), yet up to 39% of treated patients relapse with CD19(−) disease. We report that CD19(−) escape is associated with downregulation, but preservation, of targetable expression of CD20 and CD22. Accordingly, we reasoned that broadening the spectrum of CD19CAR T-cells to include both CD20 and CD22 would enable them to target CD19(−) escape BL-ALL while preserving their upfront efficacy. We created a CD19/20/22-targeting CAR T-cell by coexpressing individual CAR molecules on a single T-cell using one tricistronic transgene. CD19/20/22CAR T-cells killed CD19(−) blasts from patients who relapsed after CD19CAR T-cell therapy and CRISPR/Cas9 CD19 knockout primary BL-ALL both in vitro and in an animal model, while CD19CAR T-cells were ineffective. At the subcellular level, CD19/20/22CAR T-cells formed dense immune synapses with target cells that mediated effective cytolytic complex formation, were efficient serial killers in single-cell tracking studies, and were as efficacious as CD19CAR T-cells against primary CD19(+) disease. In conclusion, independent of CD19 expression, CD19/20/22CAR T-cells could be used as salvage or front-line CAR therapy for patients with recalcitrant disease.

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Chain Graph Models to Elicit the Structure of a Bayesian Network

The Scientific World JOURNAL ◽

10.1155/2014/749150 ◽

2014 ◽

Vol 2014 ◽

pp. 1-12 ◽

Cited By ~ 1

Author(s):

Federico M. Stefanini

Keyword(s):

Graphical Models ◽

Bayesian Methods ◽

Prior Distribution ◽

Graph Model ◽

Structural Features ◽

Chain Graph ◽

Graph Models ◽

Degree Of Belief ◽

Statistical Background ◽

A Chain

Bayesian networks are possibly the most successful graphical models to build decision support systems. Building the structure of large networks is still a challenging task, but Bayesian methods are particularly suited to exploit experts’ degree of belief in a quantitative way while learning the network structure from data. In this paper details are provided about how to build a prior distribution on the space of network structures by eliciting a chain graph model on structural reference features. Several structural features expected to be often useful during the elicitation are described. The statistical background needed to effectively use this approach is summarized, and some potential pitfalls are illustrated. Finally, a few seminal contributions from the literature are reformulated in terms of structural features.

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Capturing T Lymphocytes’ Dynamic Interactions With Human Neural Cells Using Time-Lapse Microscopy

Frontiers in Immunology ◽

10.3389/fimmu.2021.668483 ◽

2021 ◽

Vol 12 ◽

Author(s):

Florent Lemaître ◽

Ana Carmena Moratalla ◽

Negar Farzam-kia ◽

Yves Carpentier Solorio ◽

Olivier Tastet ◽

...

Keyword(s):

T Cell ◽

T Lymphocytes ◽

T Lymphocyte ◽

Cell Tracking ◽

Time Lapse ◽

Neural Cells ◽

Visual Interpretation ◽

Time Lapse Microscopy ◽

Multiple Variables

To fully perform their functions, T lymphocytes migrate within organs’ parenchyma and interact with local cells. Infiltration of T lymphocytes within the central nervous system (CNS) is associated with numerous neurodegenerative disorders. Nevertheless, how these immune cells communicate and respond to neural cells remains unresolved. To investigate the behavior of T lymphocytes that reach the CNS, we have established an in vitro co-culture model and analyzed the spatiotemporal interactions between human activated CD8+ T lymphocytes and primary human astrocytes and neurons using time-lapse microscopy. By combining multiple variables extracted from individual CD8+ T cell tracking, we show that CD8+ T lymphocytes adopt a more motile and exploratory behavior upon interacting with astrocytes than with neurons. Pretreatment of astrocytes or neurons with IL-1β to mimic in vivo inflammation significantly increases CD8+ T lymphocyte motility. Using visual interpretation and analysis of numerical variables extracted from CD8+ T cell tracking, we identified four distinct CD8+ T lymphocyte behaviors: scanning, dancing, poking and round. IL-1β-pretreatment significantly increases the proportion of scanning CD8+ T lymphocytes, which are characterized by active exploration, and reduces the proportion of round CD8+ T lymphocytes, which are less active. Blocking MHC class I on astrocytes significantly diminishes the proportion of poking CD8+ T lymphocytes, which exhibit synapse-like interactions. Lastly, our co-culture time-lapse model is easily adaptable and sufficiently sensitive and powerful to characterize and quantify spatiotemporal interactions between human T lymphocytes and primary human cells in different conditions while preserving viability of fragile cells such as neurons and astrocytes.

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