Chimeric antigen receptors (CARs) are receptors for antigen that direct potent immune responses. Tumor escape associated with low target antigen expression is emerging as one potential limitation of ...their efficacy. Here we edit the TRAC locus in human peripheral blood T cells to engage cell-surface targets through their T cell receptor-CD3 complex reconfigured to utilize the same immunoglobulin heavy and light chains as a matched CAR. We demonstrate that these HLA-independent T cell receptors (HIT receptors) consistently afford high antigen sensitivity and mediate tumor recognition beyond what CD28-based CARs, the most sensitive design to date, can provide. We demonstrate that the functional persistence of HIT T cells can be augmented by constitutive coexpression of CD80 and 4-1BBL. Finally, we validate the increased antigen sensitivity afforded by HIT receptors in xenograft mouse models of B cell leukemia and acute myeloid leukemia, targeting CD19 and CD70, respectively. Overall, HIT receptors are well suited for targeting cell surface antigens of low abundance.
High-throughput single cell methods have uncovered substantial heterogeneity in the pool of hematopoietic stem and progenitor cells (HSPCs), but how much instruction is inherited by offspring from ...their heterogeneous ancestors remains unanswered. Using a method that enables simultaneous determination of common ancestor, division number, and differentiation status of a large collection of single cells, our data revealed that murine cells that derived from a common ancestor had significant similarities in their division progression and differentiation outcomes. Although each family diversifies, the overall collection of cell types observed is composed of homogeneous families. Heterogeneity between families could be explained, in part, by differences in ancestral expression of cell-surface markers. Our analyses demonstrate that fate decision by cells are largely inherited from ancestor cells, indicating the importance of common ancestor effects. These results may have ramifications for bone marrow transplantation and leukemia, where substantial heterogeneity in HSPC behavior is observed.
Engagement of the receptor programmed cell death molecule 1 (PD-1) by its ligands PD-L1 and PD-L2 inhibits T cell-mediated immune responses. Blocking such signaling provides the clinical effects of ...PD-1-targeted immunotherapy. Here, we investigated the mechanisms underlying PD-1-mediated inhibition. Because dynamic actin remodeling is crucial for T cell functions, we characterized the effects of PD-1 engagement on actin remodeling at the immunological synapse, the interface between a T cell and an antigen-presenting cell (APC) or target cell. We used microscopy to analyze the formation of immunological synapses between PD-1
Jurkat cells or primary human CD8
cytotoxic T cells and APCs that presented T cell-activating antibodies and were either positive or negative for PD-L1. PD-1 binding to PD-L1 inhibited T cell spreading induced by antibody-mediated activation, which was characterized by the absence of the F-actin-dense distal lamellipodial network at the immunological synapse and the Arp2/3 complex, which mediates branched actin formation. PD-1-induced inhibition of actin remodeling also prevented the characteristic deformation of T cells that contact APCs and the release of cytotoxic granules. We showed that the effects of PD-1 on actin remodeling did not require its tyrosine-based signaling motifs, which are thought to mediate the co-inhibitory effects of PD-1. Our study highlights a previously unappreciated mechanism of PD-1-mediated suppression of T cell activity, which depends on the regulation of actin cytoskeleton dynamics in a signaling motif-independent manner.
In this issue of JEM, Xiaozheng Xu et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20221391) report that the inhibitory protein CTLA4 internalizes in cis the B7 stimulatory molecules previously ..."gnawed" by T cells from antigen-presenting cells (APCs) and in doing so prevents stimulatory T-T interactions.
T-cell activation induces a metabolic switch generating energy for proliferation, survival, and functions. We used noninvasive label-free two-photon fluorescence lifetime microscopy (2P-FLIM) to map ...the spatial and temporal dynamics of the metabolic NAD(P)H co-enzyme during T lymphocyte activation. This provides a readout of the OXPHOS and glycolysis rates at a single-cell level. Analyzes were performed in the CD4+ leukemic T cell line Jurkat, and in human CD4+ primary T cells. Cells were activated on glass surfaces coated with activating antibodies mimicking immune synapse formation. Comparing the fraction of bound NAD(P)H between resting and activated T cells, we show that T-cell activation induces a rapid switch toward glycolysis. This occurs after 10 min and remains stable for one hour. Three-dimensional analyzes revealed that the intracellular distribution of fraction of bound NAD(P)H increases at the immune synapse in activated cells. Finally, we show that fraction of bound NAD(P)H tends to negatively correlate with spreading of activated T cells, suggesting a link between actin remodeling and metabolic changes. This study highlights that 2P-FLIM measurement of fraction of bound NAD(P)H is well suited to follow a fast metabolic switch in three dimensions, in single T lymphocytes with subcellular resolution.
BackgroundChimeric antigen receptors (CARs) engage antigen independently of HLA and enable sustained T cell proliferation when they are endowed with both activating and costimulatory functions. While ...remission rates have been noticeably elevated in numerous clinical trials targeting CD19, CD22 or BCMA, relapses are common. One of the several underlying relapse mechanisms is antigen escape, which refers to a relapsing tumor that is either negative for the targeted antigen or expresses the latter at a low level. Failure to eliminate antigen-low tumors raises questions about the sensitivity of CARs and the minimum antigen density that is required for effective tumor eradication. Unlike CARs, TCRs engage antigen in an HLA-dependent manner, and they do so with high sensitivity. We hypothesized that a TCR/CD3 complex containing the same heavy and light immunoglobulin chains as a CAR will display increased sensitivity to the target antigen.MethodsWe edited the TRAC locus in human primary T cells to establish a novel antigen receptor structure, termed HLA-independent TCR or HIT receptor, by incorporating into the TCR/CD3 complex the same heavy and light chains as those of a corresponding CAR. We assessed their antigen sensitivity against a panel of cell lines expressing different antigen levels, analyzing their cytotoxicity, cytokine secretion, signaling response and degranulation activity. HIT and CAR T cells were further evaluated for their anti-tumor response using established ALL and AML mouse models.ResultsCD19-TRAC-HIT and CD19-TRAC-CAR T cells lysed wild-type NALM6 (~27,000 CD19 molecules) and NALM6 variants with 100-fold less CD19. As CD19 levels decreased further, CAR T cells no longer killed their target, in contrast to HIT T cells. HIT T cells showed increased expression of IFN-gamma, IL-2 and TNF-alpha upon exposure to NALM6 cells expressing ~20 CD19 molecules per cell, compared to CAR T cells. This increased sensitivity of HIT receptors correlated to their greater signaling response, upon exposure to the low-antigen-density NALM6. Phospho-proteomic analyses further confirmed this increased response of HIT T cells to low antigen levels. Altogether, these results confirm that HIT receptors endow T cells with greater antigen sensitivity than canonical CARs. We further showed that HIT T cells have higher in vivo anti-tumor activity compared to CAR T cells in mice bearing low-antigen-density ALL or AML.ConclusionsHIT receptors consistently afford high antigen sensitivity and mediate tumor recognition beyond what current CARs can provide. HIT receptors open new prospects for targeting cell surface antigens of low abundance.Ethics ApprovalEight- to 12-week-old NOD/SCID/IL-2Rgamma-null (NSG) male mice (Jackson Laboratory) were used under a protocol approved by the MSKCC Institutional Animal Care and Use Committee.
Mechanobiology of antigen‐induced T cell arrest Chabaud, Mélanie; Paillon, Noémie; Gaus, Katharina ...
Biology of the cell,
July 2020, 2020-Jul, 2020-07-00, 20200701, Letnik:
112, Številka:
7
Journal Article
Recenzirano
To mount an immune response, T cells must first find rare antigens present at the surface of antigen‐presenting cells (APCs). They achieve this by migrating rapidly through the crowded space of ...tissues and constantly sampling the surface of APCs. Upon antigen recognition, T cells decelerate and polarise towards the APC, ultimately forming a specialised interface known as the immunological synapse. These conjugates form as the result of the interaction between pairs of receptors/ligands that are under mechanical stress due to the continuously reorganising cell cytoskeleton. In this review, we discuss the involvement of mechanical forces during antigen recognition by migrating T cells. We will explore this question from a conceptual and technical perspective, with the aim of providing new insights into the emerging field of mechanobiology.
Review: Antigen recognition by T cells leads to their arrest on the surface of antigen‐presenting cells. This event is associated with profound membrane and cytoskeletal remodelling that generate mechanical forces. This review explores our current knowledge of T cell mechanobiology and proposes additional conceptual and technical perspectives.
Background Information
We have previously observed that in response to antigenic activation, T cells produce actin‐rich protrusions that generate forces involved in T cell activation. These forces ...are influenced by the mechanical properties of antigen‐presenting cells (APCs). However, how external forces, which can be produced by APCs, influence the dynamic of the actin protrusion remains unknown. In this study, we quantitatively characterised the effects of external forces in the dynamic of the protrusion grown by activated T cells.
Results
Using a micropipette force probe, we applied controlled compressive or pulling forces on primary T lymphocytes activated by an antibody‐covered microbead, and measured the effects of these forces on the protrusion generated by T lymphocytes. We found that the application of compressive forces slightly decreased the length, the time at which the protrusion stops growing and retracts and the velocity of the protrusion formation, whereas pulling forces strongly increased these parameters. In both cases, the applied forces did not alter the time required for the T cells to start growing the protrusion (delay). Exploring the molecular events controlling the dynamic of the protrusion, we showed that inhibition of the Arp2/3 complex impaired the dynamic of the protrusion by reducing both its maximum length and its growth speed and increasing the delay to start growing. Finally, T cells developed similar protrusions in more physiological conditions, that is, when activated by an APC instead of an activating microbead.
Conclusions
Our results suggest that the formation of the force‐generating protrusion by T cells is set by an intracellular constant time and that its dynamic is sensitive to external forces. They also show that actin assembly mediated by actin‐related protein Arp2/3 complex is involved in the formation and dynamic of the protrusion.
Significance
Actin‐rich protrusions developed by T cells are sensory organelles that serve as actuators of immune surveillance. Our study shows that forces experienced by this organelle modify their dynamic suggesting that they might modify immune responses. Moreover, the quantitative aspects of our analysis should help to get insight into the molecular mechanisms involved in the formation of the protrusion.
Research article: During immunological synapse formation, T cells emit an actin‐rich protrusion that experiences forces. Here, we quantitatively characterised the effects of external forces in the dynamic of the protrusion. We found that the formation of the protrusion is set by an intracellular constant time and is mediated by the Arp2/3 complex and that its dynamic is sensitive to external forces. Our study provides key insights that help to better understand the mechanism involved in the formation of the actin protrusion emitted by the T cell.
High-throughput single-cell methods have uncovered substantial heterogeneity in the pool of hematopoietic stem and progenitor cells (HSPCs), but how much instruction is inherited by offspring from ...their heterogeneous ancestors remains unanswered. Using a method that enables simultaneous determination of common ancestor, division number, and differentiation status of a large collection of single cells, our data revealed that murine cells that derived from a common ancestor had significant similarities in their division progression and differentiation outcomes. Although each family diversifies, the overall collection of cell types observed is composed of homogeneous families. Heterogeneity between families could be explained, in part, by differences in ancestral expression of cell surface markers. Our analyses demonstrate that fate decisions of cells are largely inherited from ancestor cells, indicating the importance of common ancestor effects. These results may have ramifications for bone marrow transplantation and leukemia, where substantial heterogeneity in HSPC behavior is observed.
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