Anti-CD19 Chimeric Antigen Receptor T-Cell Therapy for Richter Transformation: An International, Multicenter, Retrospective Study.

PURPOSE: Outcomes for Richter transformation (RT) are poor with current therapies. The efficacy and safety of anti-CD19 chimeric antigen receptor T-cell therapy (CAR-T) for RT are not established. METHODS: We performed an international multicenter retrospective study of patients with RT who received CAR-T. Patient, disease, and treatment characteristics were summarized using descriptive statistics, and modeling analyses were used to determine association with progression-free survival (PFS) and overall survival (OS). PFS and OS were estimated from the date of CAR-T infusion. RESULTS: Sixty-nine patients were identified. The median age at CAR-T infusion was 64 years (range, 27-80). Patients had a median of four (range, 1-15) previous lines of therapy for CLL and/or RT, including previous Bruton tyrosine kinase inhibitor and/or BCL2 inhibitor therapy in 58 (84%) patients. The CAR-T product administered was axicabtagene ciloleucel in 44 patients (64%), tisagenlecleucel in 17 patients (25%), lisocabtagene maraleucel in seven patients (10%), and brexucabtagene autoleucel in one patient (1%). Eleven patients (16%) and 25 patients (37%) experienced grade ≥3 cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome, respectively. The overall response rate was 63%, with 46% attaining a complete response (CR). After a median follow-up of 24 months, the median PFS was 4.7 months (95% CI, 2.0 to 6.9); the 2-year PFS was 29% (95% CI, 18 to 41). The median OS was 8.5 months (95% CI, 5.1 to 25.4); the 2-year OS was 38% (95% CI, 26 to 50). The median duration of response was 27.6 months (95% CI, 14.5 to not reached) for patients achieving CR. CONCLUSION: CAR-T demonstrates clinical efficacy for patients with RT.

Intrinsically determined turnover underlies broad heterogeneity in plasma-cell lifespan.

Antibodies produced by antibody-secreting plasma cells (ASCs) underlie multiple forms of long-lasting immunity. Here we examined the mechanisms regulating ASC turnover and persistence using a genetic reporter to time-stamp ASCs. This approach revealed ASC lifespans as heterogeneous and falling on a continuum, with only a small fraction surviving for >60 days. ASC longevity past 60 days was independent of isotype but correlated with a phenotype that developed progressively and ultimately associated with an underlying "long-lived" ASC (LL ASC)-enriched transcriptional program. While some of the differences between LL ASCs and other ASCs appeared to be acquired with age, other features were shared with some younger ASCs, such as high CD138 and CD93. Turnover was unaffected by altered ASC production, arguing against competition for niches as a major driver of turnover. Thus, ASC turnover is set by intrinsic lifespan limits, with steady-state population dynamics governed by niche vacancy rather than displacement.

In vivo PIWI slicing in mouse testes deviates from rules established in vitro.

Argonautes are small RNA-binding proteins, with some having small RNA-guided endonuclease (slicer) activity that cleaves target nucleic acids. One cardinal rule that is structurally defined is the inability of slicers to cleave target RNAs when nucleotide mismatches exist between the paired small RNA and the target at the cleavage site. Animal-specific PIWI clade Argonautes associate with PIWI-interacting RNAs (piRNAs) to silence transposable elements in the gonads, and this is essential for fertility. We previously demonstrated that purified endogenous mouse MIWI fails to cleave mismatched targets in vitro. Surprisingly, here we find using knock-in mouse models that target sites with cleavage-site mismatches at the 10th and 11th piRNA nucleotides are precisely sliced in vivo. This is identical to the slicing outcome in knock-in mice where targets are base-paired perfectly with the piRNA. Additionally, we find that pachytene piRNA-guided slicing in both these situations failed to initiate phased piRNA production from the specific target mRNA we studied. Instead, the two slicer cleavage fragments were retained in PIWI proteins as pre-piRNA and 17-19 nt by-product fragments. Our results indicate that PIWI slicing rules established in vitro are not respected in vivo, and that all targets of PIWI slicing are not substrates for piRNA biogenesis.

Intermitochondrial cement (IMC) harbors piRNA biogenesis machinery and exonuclease domain-containing proteins EXD1 and EXD2 in mouse spermatocytes.

BACKGROUND: Germ granules are large cytoplasmic ribonucleoprotein complexes that emerge in the germline to participate in RNA regulation. The two most prominent germ granules are the intermitochondrial cement (IMC) in meiotic spermatocytes and the chromatoid body (CB) in haploid round spermatids, both functionally linked to the PIWI-interacting RNA (piRNA) pathway. AIMS: In this study, we clarified the IMC function by identifying proteins that form complexes with a well-known IMC protein PIWIL2/MILI in the mouse testis. RESULTS: The PIWIL2 interactome included several proteins with known functions in piRNA biogenesis. We further characterized the expression and localization of two of the identified proteins, Exonuclease 3'-5' domain-containing proteins EXD1 and EXD2, and confirmed their localization to the IMC. We showed that EXD2 interacts with PIWIL2, and that the mutation of Exd2 exonuclease domain in mice induces misregulation of piRNA levels originating from specific pachytene piRNA clusters, but does not disrupt male fertility. CONCLUSION: Altogether, this study highlights the central role of the IMC as a platform for piRNA biogenesis, and suggests that EXD1 and EXD2 function in the IMC-mediated RNA regulation in postnatal male germ cells.

Long-lived plasma cells accumulate in the bone marrow at a constant rate from early in an immune response.

Vaccines work largely by generating long-lived plasma cells (LLPCs), but knowledge of how such cells are recruited is sparse. Although it is clear that LLPCs preferentially originate in germinal centers (GCs) and relocate to survival niches in bone marrow where they can persist for decades, the issues of the timing of LLPC recruitment and the basis of their retention remain uncertain. Here, using a genetic timestamping system in mice, we show that persistent PCs accrue in bone marrow at an approximately constant rate of one cell per hour over a period spanning several weeks after a single immunization with a model antigen. Affinity-based selection was evident in persisting PCs, reflecting a relative and dynamic rather than absolute affinity threshold as evidenced by the changing pattern of VH gene somatic mutations conveying increased affinity for antigen. We conclude that the life span of persistent, antigen-specific PCs is in part intrinsic, preprogrammed, and varied and that their final number is related to the duration of the response in a predictable way. This implies that modulating vaccines to extend the duration of the GC reaction will enhance antibody-mediated protective immunity.

NKG7 Enhances CD8+ T Cell Synapse Efficiency to Limit Inflammation.

Cytotoxic lymphocytes are essential for anti-tumor immunity, and for effective responses to cancer immunotherapy. Natural killer cell granule protein 7 (NKG7) is expressed at high levels in cytotoxic lymphocytes infiltrating tumors from patients treated with immunotherapy, but until recently, the role of this protein in cytotoxic lymphocyte function was largely unknown. Unexpectedly, we found that highly CD8+ T cell-immunogenic murine colon carcinoma (MC38-OVA) tumors grew at an equal rate in Nkg7+/+ and Nkg7-/- littermate mice, suggesting NKG7 may not be necessary for effective CD8+ T cell anti-tumor activity. Mechanistically, we found that deletion of NKG7 reduces the ability of CD8+ T cells to degranulate and kill target cells in vitro. However, as a result of inefficient cytotoxic activity, NKG7 deficient T cells form a prolonged immune synapse with tumor cells, resulting in increased secretion of inflammatory cytokines, including tumor necrosis factor alpha (TNF). By deleting the TNF receptor, TNFR1, from MC38-OVA tumors, we demonstrate that this hyper-secretion of TNF compensates for reduced synapse-mediated cytotoxic activity against MC38-OVA tumors in vivo, via increased TNF-mediated tumor cell death. Taken together, our results demonstrate that NKG7 enhances CD8+ T cell immune synapse efficiency, which may serve as a mechanism to accelerate direct cytotoxicity and limit potentially harmful inflammatory responses.

Development of autotaxin inhibitors: A series of tetrazole cinnamides.

A series of inhibitors of Autotaxin (ATX) have been developed from a high throughput screening hit, 1a, which shows an alternative binding mode to known catalytic site inhibitors. Selectivity over the hERG channel and microsomal clearance were dependent on the lipophilicity of the compounds, and this was optimised by reduction of clogD whilst maintaining high affinity ATX inhibition. Compound 15a shows good oral exposure, and concentration dependent inhibition of formation of LPA in vivo, as shown in pharmacokinetic-pharmacodynamic (PK/PD) experiments.

Germline heterozygous mutations in Nxf1 perturb RNA metabolism and trigger thrombocytopenia and lymphopenia in mice.

In eukaryotic cells, messenger RNA (mRNA) molecules are exported from the nucleus to the cytoplasm, where they are translated. The highly conserved protein nuclear RNA export factor1 (Nxf1) is an important mediator of this process. Although studies in yeast and in human cell lines have shed light on the biochemical mechanisms of Nxf1 function, its contribution to mammalian physiology is less clear. Several groups have identified recurrent NXF1 mutations in chronic lymphocytic leukemia (CLL), placing it alongside several RNA-metabolism factors (including SF3B1, XPO, RPS15) whose dysregulation is thought to contribute to CLL pathogenesis. We report here an allelic series of germline point mutations in murine Nxf1. Mice heterozygous for these loss-of-function Nxf1 mutations exhibit thrombocytopenia and lymphopenia, together with milder hematological defects. This is primarily caused by cell-intrinsic defects in the survival of platelets and peripheral lymphocytes, which are sensitized to intrinsic apoptosis. In contrast, Nxf1 mutations have almost no effect on red blood cell homeostasis. Comparative transcriptome analysis of platelets, lymphocytes, and erythrocytes from Nxf1-mutant mice shows that, in response to impaired Nxf1 function, the cytoplasmic representation of transcripts encoding regulators of RNA metabolism is altered in a unique, lineage-specific way. Thus, blood cell lineages exhibit differential requirements for Nxf1-mediated global mRNA export.

Regulatory T Cells Suppress Effector T Cell Proliferation by Limiting Division Destiny.

Understanding how the strength of an effector T cell response is regulated is a fundamental problem in immunology with implications for immunity to pathogens, autoimmunity, and immunotherapy. The initial magnitude of the T cell response is determined by the sum of independent signals from antigen, co-stimulation and cytokines. By applying quantitative methods, the contribution of each signal to the number of divisions T cells undergo (division destiny) can be measured, and the resultant exponential increase in response magnitude accurately calculated. CD4+CD25+Foxp3+ regulatory T cells suppress self-reactive T cell responses and limit pathogen-directed immune responses before bystander damage occurs. Using a quantitative modeling framework to measure T cell signal integration and response, we show that Tregs modulate division destiny, rather than directly increasing the rate of death or delaying interdivision times. The quantitative effect of Tregs could be mimicked by modulating the availability of stimulatory co-stimuli and cytokines or through the addition of inhibitory signals. Thus, our analysis illustrates the primary effect of Tregs on the magnitude of effector T cell responses is mediated by modifying division destiny of responding cell populations.

Sequential deposition of microdroplets on patterned surfaces.

We use a combination of experiments and numerical modelling to investigate the influence of physico-chemical-patterned substrates on the spreading of fluid deposited as a partially overlapping sequence of droplets via inkjet printing. Our investigation is motivated by the manufacture of polymeric organic light-emitting-diode displays, where the substrate is textured with a regular array of shallow recessed regions (pixels) that are highly wetting compared to the remainder of the substrate. We examine the roles of topography and wettability patterning separately and in combination. On a substrate with uniform wettability, we find that the presence of bounding side walls enhances the local spreading and facilitates fluid coverage of the entire recessed region, but containment within the pixel is not guaranteed. In contrast, wettability patterning alone leads to robust containment of the fluid within the wetting region, but fluid coverage is reduced in the absence of side walls. Our theoretical calculations use a simplified numerical model of fluid redistribution via purely capillary effects, augmented by a Cox-Voinov spreading law. The neglect of fluid viscosity in this model means that, after an initial period of agreement, the predicted evolution is faster than in the experiments. Nonetheless, the simplified model achieves excellent predictions both for the liquid morphologies and for the conditions required for successful pixel filling on substrates with topographical and wettability variations.

Development of autotaxin inhibitors: A series of zinc binding triazoles.

A series of inhibitors of Autotaxin (ATX) has been developed using the binding mode of known inhibitor, PF-8380, as a template. Replacement of the benzoxazolone with a triazole zinc-binding motif reduced crystallinity and improved solubility relative to PF-8380. Modification of the linker region removed hERG activity and led to compound 12 - a selective, high affinity, orally-bioavailable inhibitor of ATX. Compound 12 concentration-dependently inhibits autotaxin and formation of LPA in vivo, as shown in pharmacokinetic-pharmacodynamic experiments.

Selective inhibition of histamine-evoked Ca2+ signals by compartmentalized cAMP in human bronchial airway smooth muscle cells.

Intracellular Ca2+ and cAMP typically cause opposing effects on airway smooth muscle contraction. Receptors that stimulate these pathways are therapeutic targets in asthma and chronic obstructive pulmonary disease. However, the interactions between different G protein-coupled receptors (GPCRs) that evoke cAMP and Ca2+ signals in human bronchial airway smooth muscle cells (hBASMCs) are poorly understood. We measured Ca2+ signals in cultures of fluo-4-loaded hBASMCs alongside measurements of intracellular cAMP using mass spectrometry or [3H]-adenine labeling. Interactions between the signaling pathways were examined using selective ligands of GPCRs, and inhibitors of Ca2+ and cAMP signaling pathways. Histamine stimulated Ca2+ release through inositol 1,4,5-trisphosphate (IP3) receptors in hBASMCs. ß2-adrenoceptors, through cAMP and protein kinase A (PKA), substantially inhibited histamine-evoked Ca2+ signals. Responses to other Ca2+-mobilizing stimuli were unaffected by cAMP (carbachol and bradykinin) or minimally affected (lysophosphatidic acid). Prostaglandin E2 (PGE2), through EP2 and EP4 receptors, stimulated formation of cAMP and inhibited histamine-evoked Ca2+ signals. There was no consistent relationship between the inhibition of Ca2+ signals and the amounts of intracellular cAMP produced by different stimuli. We conclude that ß-adrenoceptors, EP2 and EP4 receptors, through cAMP and PKA, selectively inhibit Ca2+ signals evoked by histamine in hBASMCs, suggesting that PKA inhibits an early step in H1 receptor signaling. Local delivery of cAMP within hyperactive signaling junctions mediates the inhibition.

Endogenous lysophosphatidic acid (LPA1 ) receptor agonists demonstrate ligand bias between calcium and ERK signalling pathways in human lung fibroblasts.

BACKGROUND AND PURPOSE: Human lung fibroblasts (HLF) express high levels of the LPA1 receptor, a GPCR that responds to the endogenous lipid mediator, lysophosphatidic acid (LPA). Several molecular species or analogues of LPA exist and have been detected in biological fluids such as serum and plasma. The most widely expressed of the LPA receptor family is the LPA1 receptor, which predominantly couples to Gq/11 , Gi/o and G12/13 proteins. This promiscuity of coupling raises the possibility that some of the LPA analogues may bias the LPA1 receptor towards one signalling pathway over another. EXPERIMENTAL APPROACH: Here, we have explored the signalling profiles of a range of LPA analogues in HLF that endogenously express the LPA1 receptor. HLF were treated with LPA analogues and receptor activation monitored via calcium mobilization and ERK phosphorylation. KEY RESULTS: These analyses demonstrated that the 16:0, 17:0, 18:2 and C18:1 LPA analogues appear to exhibit ligand bias between ERK phosphorylation and calcium mobilization when compared with 18:1 LPA, one of the most abundant forms of LPA that has been found in human plasma. CONCLUSION AND IMPLICATIONS: The importance of LPA as a key signalling molecule is shown by its widespread occurrence in biological fluids and its association with disease conditions such as fibrosis and cancer. These findings have important, as yet unexplored, implications for the (patho-) physiological signalling of the LPA1 receptor, as it may be influenced not only by the concentration of endogenous ligand but the isoform as well.

Stochastic Measurement Models for Quantifying Lymphocyte Responses Using Flow Cytometry.

Adaptive immune responses are complex dynamic processes whereby B and T cells undergo division and differentiation triggered by pathogenic stimuli. Deregulation of the response can lead to severe consequences for the host organism ranging from immune deficiencies to autoimmunity. Tracking cell division and differentiation by flow cytometry using fluorescent probes is a major method for measuring progression of lymphocyte responses, both in vitro and in vivo. In turn, mathematical modeling of cell numbers derived from such measurements has led to significant biological discoveries, and plays an increasingly important role in lymphocyte research. Fitting an appropriate parameterized model to such data is the goal of these studies but significant challenges are presented by the variability in measurements. This variation results from the sum of experimental noise and intrinsic probabilistic differences in cells and is difficult to characterize analytically. Current model fitting methods adopt different simplifying assumptions to describe the distribution of such measurements and these assumptions have not been tested directly. To help inform the choice and application of appropriate methods of model fitting to such data we studied the errors associated with flow cytometry measurements from a wide variety of experiments. We found that the mean and variance of the noise were related by a power law with an exponent between 1.3 and 1.8 for different datasets. This violated the assumptions inherent to commonly used least squares, linear variance scaling and log-transformation based methods. As a result of these findings we propose a new measurement model that we justify both theoretically, from the maximum entropy standpoint, and empirically using collected data. Our evaluation suggests that the new model can be reliably used for model fitting across a variety of conditions. Our work provides a foundation for modeling measurements in flow cytometry experiments thus facilitating progress in quantitative studies of lymphocyte responses.

T cell signaling. Antigen affinity, costimulation, and cytokine inputs sum linearly to amplify T cell expansion.

T cell responses are initiated by antigen and promoted by a range of costimulatory signals. Understanding how T cells integrate alternative signal combinations and make decisions affecting immune response strength or tolerance poses a considerable theoretical challenge. Here, we report that T cell receptor (TCR) and costimulatory signals imprint an early, cell-intrinsic, division fate, whereby cells effectively count through generations before returning automatically to a quiescent state. This autonomous program can be extended by cytokines. Signals from the TCR, costimulatory receptors, and cytokines add together using a linear division calculus, allowing the strength of a T cell response to be predicted from the sum of the underlying signal components. These data resolve a long-standing costimulation paradox and provide a quantitative paradigm for therapeutically manipulating immune response strength.