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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. HLA-independent T cell receptors, in which the heavy and light chains of a chimeric antigen receptor are incorporated into the endogenous T cell receptor locus, are more effective than CD28-based chimeric antigen receptors at targeting tumors with low antigen expression.

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.

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.

The protein of PD-1 receptor is induced during T cell activation, and its ligand is present on many tissue-forming cells, such as epithelial, endothelial, but also professional antigen presenting cells. The interaction between PD-1 and PD-L1 inhibits effector functions of T cells, thus preventing the development of autoimmune diseases. In some disease contexts, such as cancer or chronic infections, the persistent presence of an antigen can result in prolonged expression of PD-1 and lead to an exhausted phenotype of T cells that prevents them from eliminating the pathogen. The PD-1/PD-L1 interaction is then deleterious and leads to tumor progression or prevents resolution of a chronic viral infection. Numerous studies have proposed the PD-1/PD-L1 interaction as a therapeutic target for cancer treatment, and monoclonal antibodies preventing this binding have shown significant clinical efficacy. However, these treatments are not effective in some patients, and it is therefore essential to better understand the mechanisms of PD-1-mediated T cell inactivation.An essential mechanism for efficient T cell activation is the formation of an immunological synapse with an antigen presenting cell. Thus, the objective of my thesis was to characterize the impact of the PD-1/PD-L1 interaction on the formation of an immunological synapse. Specifically, I addressed the following questions:1. What are the effects of PD-1 on actin cytoskeleton remodeling at the immunological synapse? My work has shown that PD-1 inhibits actin remodeling at the immunological synapse. Indeed, it prevents the formation of a dense ring-shaped actin meshwork at the periphery of the contact, and a hypodense zone in the center. The PD-1/PD-L1 interaction also prevents the morphological changes of T cells at the immunological synapse. The results suggest that PD-1/PD-L1 interaction alters the activity of the Arp2/3 complex initiating branched actin polymerization.2. What are the implications of these alterations on the effector functions of T cells, in particular on the cytotoxicity of CD8+ T cells? The absence of the hypodense area in the center of the contact causes a physical barrier preventing the secretion of cytolytic granules. It can be restored by the pharmacological opening of the actin meshwork at the synapse. Thus, the effects of PD-1 on actin may bring a new explanation for the inability of T cells to lyse target cells in the presence of PD-1/PD-L1.3. Are these alterations dependent on PD-1 signaling motifs? The inhibitory effects of PD-1 are generally attributed to its ability to recruit the phosphatase SHP-2 which dephosphorylates key proteins in the TCR and CD28 signaling pathways. By studying the effects of PD-1 mutated on the motifs that recruit SHP-2, I was able to demonstrate that the defects in actin remodeling induced by PD-1 are independent of the signaling motifs. This result is the first report of an SHP-2-independent inhibitory effect of PD-1.Thus, we report a novel inhibitory effect of PD-1 on T cell actin remodeling at the immunological synapse. This PD-1 inhibitory effect results in an absence of actin clearance at the center of the contact zone, which prevents the secretion of cytolytic granules, and thus the cytotoxic capacity of T cells. This inhibition appears to be independent of PD-1 signaling motifs that allow the recruitment of the SHP-2 phosphatase.

Naïve T cell activation requires not only antigen recognition through the TCR but also a co-stimulatory signal, mainly provided by CD28. Here, using a T cell–specific conditional knockout (cKO) model, we identify the microtubule-affinity kinase 2 (MARK2) as a key intracellular checkpoint that limits CD28-mediated co-stimulation. In vivo, MARK2 deficiency promotes the development of central memory T cells, enhances basal glycolysis activity in naïve CD8 T cells, and leads to the development of systemic autoimmunity in aged mice. In MARK2-deficient CD8 T cells, TCR engagement alone drives sustained proliferation, cytokine production, and glycolysis, processes that normally require CD28 co-stimulation. Single-cell transcriptomic analysis reveals that MARK2 regulates the expression of genes involved in CD28 signaling and metabolic switch. We show that MARK2 restrains the PI3K–AKT–mTORC1 pathway by limiting CD28-driven transcriptional and metabolic programs. Mechanistically, we demonstrate that MARK2 phosphorylates CREB regulated transcription coactivator 2 (CRTC2) and suppresses CREB-mediated transcription and mTOR activation, whereas CD28 engagement lifts this inhibition. Together, our results redefine the role of CD28 that not only amplifies TCR signaling but also relieves a MARK2-dependent inhibitory signal. This work provides new insights into T cell activation, metabolism and immune tolerance with potential implications for immunotherapeutic strategies in cancer and autoimmunity.

T cell activation induces a metabolic switch generating energy required for proliferation, survival, and fueling their functions. Thus, it is essential to monitor metabolism associated to subcellular functional and structural changes. We used non-invasive label-free two-photon fluorescence lifetime microscopy (2P-FLIM) to map the spatial and temporal dynamics of the metabolic NADH co-enzyme during T lymphocyte activation. 2P-FLIM measurements of the protein-bound and free NADH ratios provides a readout of the redox state (NAD+/ NADH) of the cells, and thus of their OXPHOS and glycolysis rates. Using this method, we followed the dynamics of fraction of bound NADH (fb NADH) in live single cells. Comparing fb NADH between resting and activated T cells, we show that T cell activation induces a rapid switch toward glycolysis. The switch takes only 10 minutes and remains stable for at least one hour. Three-dimensional (3D) analysis revealed that the intracellular distribution of fb NADH is symmetrically distributed in resting cells, whereas increases at the contact zone in activated cells. Finally, we show that fb NADH negatively correlates with spreading of activated T cells, suggesting a link between actin remodeling and metabolic changes. This study shows that 2P-FLIM measurement of fb NADH is well suited to follow a fast metabolic switch in 3D, in single T lymphocytes with subcellular resolution.Competing Interest StatementThe authors have declared no competing interest.Footnotes* https://zenodo.org/badge/DOI/10.5281/zenodo.7473905.svg* https://zenodo.org/badge/DOI/10.5281/zenodo.7474053.svg

The advent of high throughput single cell methods such as scRNA-seq has uncovered substantial heterogeneity in the pool of hematopoietic stem and progenitor cells (HSPCs). A significant issue is how to reconcile those findings with the standard model of hematopoietic development, and a fundamental question is how much instruction is inherited by offspring from their ancestors. To address this, we further developed a high-throughput method that enables simultaneously determination of common ancestor, generation, and differentiation status of a large collection of single cells. Data from it revealed that while there is substantial population-level heterogeneity, cells that derived from a common ancestor were highly concordant in their division progression and share similar differentiation outcomes, revealing significant familial effects on both division and differentiation. Although each family diversifies to some extent, the overall collection of cell types observed in a population is largely composed of homogeneous families from heterogeneous ancestors. Heterogeneity between families could be explained, in part, by differences in ancestral expression of cell-surface markers that are used for phenotypic HSPC identification: CD48, SCA-1, c-kit and Flt3. These data call for a revision of the fundamental model of haematopoiesis from a single tree to an ensemble of trees from distinct ancestors where common ancestor effect must be considered. As HSPCs are cultured in the clinic before bone marrow transplantation, our results suggest that the broad range of engraftment and proliferation capacities of HSPCs could be consequences of the heterogeneity in their engrafted families, and altered culture conditions might reduce heterogeneity between families, possibly improving transplantation outcomes.