Leukemia-intrinsic determinants of CAR-T response revealed by iterative in vivo genome-wide CRISPR screening.

CAR-T therapy is a promising, novel treatment modality for B-cell malignancies and yet many patients relapse through a variety of means, including loss of CAR-T cells and antigen escape. To investigate leukemia-intrinsic CAR-T resistance mechanisms, we performed genome-wide CRISPR-Cas9 loss-of-function screens in an immunocompetent murine model of B-cell acute lymphoblastic leukemia (B-ALL) utilizing a modular guide RNA library. We identified IFNγR/JAK/STAT signaling and components of antigen processing and presentation pathway as key mediators of resistance to CAR-T therapy in vivo; intriguingly, loss of this pathway yielded the opposite effect in vitro (sensitized leukemia to CAR-T cells). Transcriptional characterization of this model demonstrated upregulation of these pathways in tumors relapsed after CAR-T treatment, and functional studies showed a surprising role for natural killer (NK) cells in engaging this resistance program. Finally, examination of data from B-ALL patients treated with CAR-T revealed an association between poor outcomes and increased expression of JAK/STAT and MHC-I in leukemia cells. Overall, our data identify an unexpected mechanism of resistance to CAR-T therapy in which tumor cell interaction with the in vivo tumor microenvironment, including NK cells, induces expression of an adaptive, therapy-induced, T-cell resistance program in tumor cells.

LFA-1 and kindlin-3 enable the collaborative transport of SLP-76 microclusters by myosin and dynein motors.

Integrin engagement within the immune synapse enhances T cell activation, but our understanding of this process is incomplete. In response to T cell receptor (TCR) ligation, SLP-76 (LCP2), ADAP (FYB1) and SKAP55 (SKAP1) are recruited into microclusters and activate integrins via the effectors talin-1 and kindlin-3 (FERMT3). We postulated that integrins influence the centripetal transport and signaling of SLP-76 microclusters via these linkages. We show that contractile myosin filaments surround and are co-transported with SLP-76 microclusters, and that TCR ligand density governs the centripetal movement of both structures. Centripetal transport requires formin activity, actomyosin contraction, microtubule integrity and dynein motor function. Although immobilized VLA-4 (α4ß1 integrin) and LFA-1 (αLß2 integrin) ligands arrest the centripetal movement of SLP-76 microclusters and myosin filaments, VLA-4 acts distally, while LFA-1 acts in the lamellum. Integrin ß2, kindlin-3 and zyxin are required for complete centripetal transport, while integrin ß1 and talin-1 are not. CD69 upregulation is similarly dependent on integrin ß2, kindlin-3 and zyxin, but not talin-1. These findings highlight the integration of cytoskeletal systems within the immune synapse and reveal extracellular ligand-independent roles for LFA-1 and kindlin-3. This article has an associated First Person interview with the first author of the paper.

cFLIPL protects macrophages from LPS-induced pyroptosis via inhibition of complex II formation.

Cell death and inflammation are interdependent host responses to infection. During pyroptotic cell death, interleukin-1ß (IL-1ß) release occurs through caspase-1 and caspase-11-mediated gasdermin D pore formation. In vivo, responses to lipopolysaccharide (LPS) result in IL-1ß secretion. In vitro, however, murine macrophages require a second "danger signal" for the inflammasome-driven maturation of IL-1ß. Recent reports have shown caspase-8-mediated pyroptosis in LPS-activated macrophages but have provided conflicting evidence regarding the release of IL-1ß under these conditions. Here, to further characterize the mechanism of LPS-induced secretion in vitro, we reveal an important role for cellular FLICE-like inhibitory protein (cFLIP) in the regulation of the inflammatory response. Specifically, we show that deficiency of the long isoform cFLIPL promotes complex II formation, driving pyroptosis, and the secretion of IL-1ß in response to LPS alone.

ADAP is an upstream regulator that precedes SLP-76 at sites of TCR engagement and stabilizes signaling microclusters.

Antigen recognition by the T cell receptor (TCR) directs the assembly of essential signaling complexes known as SLP-76 (also known as LCP2) microclusters. Here, we show that the interaction of the adhesion and degranulation-promoting adaptor protein (ADAP; also known as FYB1) with SLP-76 enables the formation of persistent microclusters and the stabilization of T cell contacts, promotes integrin-independent adhesion and enables the upregulation of CD69. By analyzing point mutants and using a novel phospho-specific antibody, we show that Y595 is essential for normal ADAP function, that virtually all tyrosine phosphorylation of ADAP is restricted to a Y595-phosphorylated (pY595) pool, and that multivalent interactions between the SLP-76 SH2 domain and its binding sites in ADAP are required to sustain ADAP phosphorylation. Although pY595 ADAP enters SLP-76 microclusters, non-phosphorylated ADAP is enriched in protrusive actin-rich structures. The pre-positioning of ADAP at the contact sites generated by these structures favors the retention of nascent SLP-76 oligomers and their assembly into persistent microclusters. Although ADAP is frequently depicted as an effector of SLP-76, our findings reveal that ADAP acts upstream of SLP-76 to convert labile, Ca2+-competent microclusters into stable adhesive junctions with enhanced signaling potential.

A DOC2 protein identified by mutational profiling is essential for apicomplexan parasite exocytosis.

Exocytosis is essential to the lytic cycle of apicomplexan parasites and required for the pathogenesis of toxoplasmosis and malaria. DOC2 proteins recruit the membrane fusion machinery required for exocytosis in a Ca(2+)-dependent fashion. Here, the phenotype of a Toxoplasma gondii conditional mutant impaired in host cell invasion and egress was pinpointed to a defect in secretion of the micronemes, an apicomplexan-specific organelle that contains adhesion proteins. Whole-genome sequencing identified the etiological point mutation in TgDOC2.1. A conditional allele of the orthologous gene engineered into Plasmodium falciparum was also defective in microneme secretion. However, the major effect was on invasion, suggesting that microneme secretion is dispensable for Plasmodium egress.

Development of a screen to dissect Toxoplasma gondii egress.

Toxoplasma gondii egress from the host cell during the lytic part of its life cycle is increasingly appreciated as a process where complex signaling mediates the parasite's response to a variety of internal and external conditions. Although several in vitro as well as physiological triggers have been identified, the molecular nature of these signaling pathways is largely unexplored. To facilitate a more comprehensive analysis of the underlying mechanism we designed a screening procedure to enrich for phenotypes with defects in induced egress. The procedure is based on in vitro induced egress and the efficient separation of intracellular from extracellular parasites. Attachment and fast reinvasion of egressed parasites are prevented by the addition of glycans, whereas PDTC is included to specifically kill the egressed, extracellular parasites. Two available mutants were used to assess the power of the screen; a temperature sensitive mutant, F-P2, with a conditionally lethal, reversible egress defect, and a mutant wherein the perforin PLP1 is knocked out displaying a constitutive, delayed egress defect. We show that mutant F-P2 can be routinely enriched over 1000-fold from a wild-type population, whereas the PLP1-KO strain cannot be enriched, fitting the underlying phenotypes. The screen efficiency facilitates the isolation of new mutants from mutagenized parasite populations. The use of various egress enhancers will allow genetic dissection of the egress signaling pathways. This is illustrated by a mutant generated using dithitotreitol as an egress enhancer, which displays a defect in dithitotreitol induced egress but not in Ca(2+) ionophore induced egress.