Applying single-cell highly multiplexed secretome proteomics to characterize immunotherapeutic products and predict clinical responses.

Genetically and phenotypically identical immune cell populations can be highly heterogenous in terms of their immune functions and protein secretion profiles. The microfluidic chip-based single-cell highly multiplexed secretome proteomics enables characterization of cellular heterogeneity of immune responses at different cellular and molecular layers. Increasing evidence has demonstrated that polyfunctional T cells that simultaneously produce 2+ proteins per cell at the single-cell level are key effector cells that contribute to the development of potent and durable cellular immunity against pathogens and cancers. The functional proteomic technology offers a wide spectrum of cellular function assessment and can uniquely define highly polyfunctional cell subsets with cytokine signatures from live individual cells. This high-dimensional single-cell analysis provides deep dissection into functional heterogeneity and helps identify predictive biomarkers and potential correlates that are crucial for immunotherapeutic product design optimization and personalized immunotherapy development to achieve better clinical outcomes.

ArhGEF12 activates Rap1A and not RhoA in human dermal microvascular endothelial cells to reduce tumor necrosis factor-induced leak.

Overwhelming inflammation in the setting of acute critical illness induces capillary leak resulting in hypovolemia, edema, tissue dysoxia, organ failure and even death. The tight junction (TJ)-dependent capillary barrier is regulated by small GTPases, but the specific regulatory molecules most active in this vascular segment under such circumstances are not well described. We set out to identify GTPase regulatory molecules specific to endothelial cells (EC) that form TJs. Transcriptional profiling of confluent monolayers of TJ-forming human dermal microvascular ECs (HDMECs) and adherens junction only forming-human umbilical vein EC (HUVECs) demonstrate ARHGEF12 is basally expressed at higher levels and is only downregulated in HDMECs by junction-disrupting tumor necrosis factor (TNF). HDMECs depleted of ArhGEF12 by siRNA demonstrate a significantly exacerbated TNF-induced decrease in trans-endothelial electrical resistance and disruption of TJ continuous staining. ArhGEF12 is established as a RhoA-GEF in HUVECs and its knock down would be expected to reduce RhoA activity and barrier disruption. Pulldown of active GEFs from HDMECs depleted of ArhGEF12 and treated with TNF show decreased GTP-bound Rap1A after four hours but increased GTP-bound RhoA after 12 h. In cell-free assays, ArhGEF12 immunoprecipitated from HDMECs is able to activate both Rap1A and RhoA, but not act on Rap2A-C, RhoB-C, or even Rap1B which shares 95% sequence identity with Rap1A. We conclude that in TJ-forming HDMECs, ArhGEF12 selectively activates Rap1A to limit capillary barrier disruption in a mechanism independent of cAMP-mediated Epac1 activation.

Dynamics of consumer-resource reaction-diffusion models: single and multiple consumer species.

A consumer-resource reaction-diffusion model with a single consumer species was proposed and experimentally studied by Zhang et al.(Ecol Lett 20:1118-1128, 2017). Analytical study on its dynamics was further performed by He et al.(J Math Biol 78:1605-1636, 2019). In this work, we completely settle the conjecture proposed by He et al.(J Math Biol 78:1605-1636, 2019) about the global dynamics of the consumer-resource model for small yield rate. We then study a multi-species consumer-resource model where all the consumer species compete with each other through depression of the limited resources by consumption and there is no direct competition between them. We show that in this case, all consumer species persist uniformly, which implies that "competition exclusion" phenomenon will never happen. We also clarify its dynamics in both homogeneous and heterogeneous environments under various circumstances.

Effector Memory-Expressing CD45RA (TEMRA) CD8+ T Cells from Kidney Transplant Recipients Exhibit Enhanced Purinergic P2X4 Receptor-Dependent Proinflammatory and Migratory Responses.

BACKGROUND: The mechanisms regulating CD8+ T cell migration to nonlymphoid tissue during inflammation have not been fully elucidated, and the migratory properties of effector memory CD8+ T cells that re-express CD45RA (TEMRA CD8+ T cells) remain unclear, despite their roles in autoimmune diseases and allotransplant rejection. METHODS: We used single-cell proteomic profiling and functional testing of CD8+ T cell subsets to characterize their effector functions and migratory properties in healthy volunteers and kidney transplant recipients with stable or humoral rejection. RESULTS: We showed that humoral rejection of a kidney allograft is associated with an accumulation of cytolytic TEMRA CD8+ T cells in blood and kidney graft biopsies. TEMRA CD8+ T cells from kidney transplant recipients exhibited enhanced migratory properties compared with effector memory (EM) CD8+ T cells, with enhanced adhesion to activated endothelium and transmigration in response to the chemokine CXCL12. CXCL12 directly triggers a purinergic P2×4 receptor-dependent proinflammatory response of TEMRA CD8+ T cells from transplant recipients. The stimulation with IL-15 promotes the CXCL12-induced migration of TEMRA and EM CD8+ T cells and promotes the generation of functional PSGL1, which interacts with the cell adhesion molecule P-selectin and adhesion of these cells to activated endothelium. Although disruption of the interaction between functional PSGL1 and P-selectin prevents the adhesion and transmigration of both TEMRA and EM CD8+ T cells, targeting VLA-4 or LFA-1 (integrins involved in T cell migration) specifically inhibited the migration of TEMRA CD8+ T cells from kidney transplant recipients. CONCLUSIONS: Our findings highlight the active role of TEMRA CD8+ T cells in humoral transplant rejection and suggest that kidney transplant recipients may benefit from therapeutics targeting these cells.

Directed movement changes coexistence outcomes in heterogeneous environments.

Understanding mechanisms of coexistence is a central topic in ecology. Mathematical analysis of models of competition between two identical species moving at different rates of symmetric diffusion in heterogeneous environments show that the slower mover excludes the faster one. The models have not been tested empirically and lack inclusions of a component of directed movement toward favourable areas. To address these gaps, we extended previous theory by explicitly including exploitable resource dynamics and directed movement. We tested the mathematical results experimentally using laboratory populations of the nematode worm, Caenorhabditis elegans. Our results not only support the previous theory that the species diffusing at a slower rate prevails in heterogeneous environments but also reveal that moderate levels of a directed movement component on top of the diffusive movement allow species to coexist. Our results broaden the theory of species coexistence in heterogeneous space and provide empirical confirmation of the mathematical predictions.

Tumor necrosis factor-induced ArhGEF10 selectively activates RhoB contributing to human microvascular endothelial cell tight junction disruption.

Capillary endothelial cells (ECs) maintain a semi-permeable barrier between the blood and tissue by forming inter-EC tight junctions (TJs), regulating selective transport of fluid and solutes. Overwhelming inflammation, as occurs in sepsis, disrupts these TJs, leading to leakage of fluid, proteins, and small molecules into the tissues. Mechanistically, disruption of capillary barrier function is mediated by small Rho-GTPases, such as RhoA, -B, and -C, which are activated by guanine nucleotide exchange factors (GEFs) and disrupted by GTPase-activating factors (GAPs). We previously reported that a mutation in a specific RhoB GAP (p190BRhoGAP) underlays a hereditary capillary leak syndrome. Tumor necrosis factor (TNF) treatment disrupts TJs in cultured human microvascular ECs, a model of capillary leak. This response requires new gene transcription and involves increased RhoB activation. However, the specific GEF that activates RhoB in capillary ECs remains unknown. Transcriptional profiling of cultured tight junction-forming human dermal microvascular endothelial cells (HDMECs) revealed that 17 GEFs were significantly induced by TNF. The function of each candidate GEF was assessed by short interfering RNA depletion and trans-endothelial electrical resistance screening. Knockown of ArhGEF10 reduced the TNF-induced loss of barrier which was phenocopied by RhoB or dual ArhGEF10/RhoB knockdown. ArhGEF10 knockdown also reduced the extent of TNF-induced RhoB activation and disruption at tight junctions. In a cell-free assay, immunoisolated ArhGEF10 selectively catalyzed nucleotide exchange to activate RhoB, but not RhoA or RhoC. We conclude ArhGEF10 is a TNF-induced RhoB-selective GEF that mediates TJ disruption and barrier loss in human capillary endothelial cells.

BMX Represses Thrombin-PAR1-Mediated Endothelial Permeability and Vascular Leakage During Early Sepsis.

RATIONALE: BMX (bone marrow kinase on the X chromosome) is highly expressed in the arterial endothelium from the embryonic stage to the adult stage in mice. It is also expressed in microvessels and the lymphatics in response to pathological stimuli. However, its role in endothelial permeability and sepsis remains unknown. OBJECTIVE: We aimed to delineate the function of BMX in thrombin-mediated endothelial permeability and the vascular leakage that occurs with sepsis in cecal ligation and puncture models. METHODS AND RESULTS: The cecal ligation and puncture model was applied to WT (wild type) and BMX-KO (BMX global knockout) mice to induce sepsis. Meanwhile, the electric cell-substrate impedance sensing assay was used to detect transendothelial electrical resistance in vitro and, the modified Miles assay was used to evaluate vascular leakage in vivo. We showed that BMX loss caused lung injury and inflammation in early cecal ligation and puncture-induced sepsis. Disruption of BMX increased thrombin-mediated permeability in mice and cultured endothelial cells by 2- to 3-fold. The expression of BMX in macrophages, neutrophils, platelets, and lung epithelial cells was undetectable compared with that in endothelial cells, indicating that endothelium dysfunction, rather than leukocyte and platelet dysfunction, was involved in vascular permeability and sepsis. Mechanistically, biochemical and cellular analyses demonstrated that BMX specifically repressed thrombin-PAR1 (protease-activated receptor-1) signaling in endothelial cells by directly phosphorylating PAR1 and promoting its internalization and deactivation. Importantly, pretreatment with the selective PAR1 antagonist SCH79797 rescued BMX loss-mediated endothelial permeability and pulmonary leakage in early cecal ligation and puncture-induced sepsis. CONCLUSIONS: Acting as a negative regulator of PAR1, BMX promotes PAR1 internalization and signal inactivation through PAR1 phosphorylation. Moreover, BMX-mediated PAR1 internalization attenuates endothelial permeability to protect vascular leakage during early sepsis.

Effect of Stressors on the Carrying Capacity of Spatially Distributed Metapopulations.

Stressors such as antibiotics, herbicides, and pollutants are becoming increasingly common in the environment. The effects of stressors on populations are typically studied in homogeneous, nonspatial settings. However, most populations in nature are spatially distributed over environmentally heterogeneous landscapes with spatially restricted dispersal. Little is known about the effects of stressors in these more realistic settings. Here, we combine laboratory experiments with novel mathematical theory to rigorously investigate how a stressor's physiological effect and spatial distribution interact with dispersal to influence population dynamics. We prove mathematically that if a stressor increases the death rate and/or simultaneously decreases the population growth rate and yield, a homogeneous distribution of the stressor leads to a lower total population size than if the same amount of the stressor was heterogeneously distributed. We experimentally test this prediction on spatially distributed populations of budding yeast (Saccharomyces cerevisiae). We find that the antibiotic cycloheximide increases the yeast death rate but reduces the growth rate and yield. Consistent with our mathematical predictions, we observe that a homogeneous spatial distribution of cycloheximide minimizes the total equilibrium size of experimental metapopulations, with the magnitude of the effect depending predictably on the dispersal rate and the geographic pattern of antibiotic heterogeneity. Our study has implications for assessing the population risk posed by pollutants, antibiotics, and global change and for the rational design of strategies for employing toxins to control pathogens and pests.

On the effects of carrying capacity and intrinsic growth rate on single and multiple species in spatially heterogeneous environments.

We first consider a diffusive logistic model of a single species in a heterogeneous environment, with two parameters, r(x) for intrinsic growth rate and K(x) for carrying capacity. When r(x) and K(x) are proportional, i.e., [Formula: see text], it is proved by Lou (J Differ Equ 223(2):400-426, 2006) that a population diffusing at any rate will reach a higher total equilibrium biomass than the population in an environment in which the same total resources are distributed homogeneously. This paper studies another case when r(x) is a constant, i.e., independent of K(x). In such case, a striking result is that for any dispersal rate, the logistic equation with spatially heterogeneous resources will always support a total population strictly smaller than the total carrying capacity at equilibrium, which is just opposite to the case [Formula: see text]. These two cases of single species models also lead to two different forms of Lotka-Volterra competition-diffusion systems. We then examine the consequences of the aforementioned difference on the two forms of competition systems. We find that the outcome of the competition in terms of the dispersal rates and spatial distributions of resources for the two forms of competition systems are again quite different. Our results indicate that in heterogeneous environments, the correlation between r(x) and K(x) has more profound impacts in population ecology than we had previously expected, at least from a mathematical point of view.

Dynamics of a consumer-resource reaction-diffusion model : Homogeneous versus heterogeneous environments.

We study the dynamics of a consumer-resource reaction-diffusion model, proposed recently by Zhang et al. (Ecol Lett 20(9):1118-1128, 2017), in both homogeneous and heterogeneous environments. For homogeneous environments we establish the global stability of constant steady states. For heterogeneous environments we study the existence and stability of positive steady states and the persistence of time-dependent solutions. Our results illustrate that for heterogeneous environments there are some parameter regions in which the resources are only partially limited in space, a unique feature which does not occur in homogeneous environments. Such difference between homogeneous and heterogeneous environments seems to be closely connected with a recent finding by Zhang et al. (2017), which says that in consumer-resource models, homogeneously distributed resources could support higher population abundance than heterogeneously distributed resources. This is opposite to the prediction by Lou (J Differ Equ 223(2):400-426, 2006. https://doi.org/10.1016/j.jde.2005.05.010 ) for logistic-type models. For both small and high yield rates, we also show that when a consumer exists in a region with a heterogeneously distributed input of exploitable renewed limiting resources, the total population abundance at equilibrium can reach a greater abundance when it diffuses than when it does not. In contrast, such phenomenon may fail for intermediate yield rates.

Reduced high-frequency motor neuron firing, EMG fractionation, and gait variability in awake walking ALS mice.

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease prominently featuring motor neuron (MN) loss and paralysis. A recent study using whole-cell patch clamp recording of MNs in acute spinal cord slices from symptomatic adult ALS mice showed that the fastest firing MNs are preferentially lost. To measure the in vivo effects of such loss, awake symptomatic-stage ALS mice performing self-initiated walking on a wheel were studied. Both single-unit extracellular recordings within spinal cord MN pools for lower leg flexor and extensor muscles and the electromyograms (EMGs) of the corresponding muscles were recorded. In the ALS mice, we observed absent or truncated high-frequency firing of MNs at the appropriate time in the step cycle and step-to-step variability of the EMG, as well as flexor-extensor coactivation. In turn, kinematic analysis of walking showed step-to-step variability of gait. At the MN level, the higher frequencies absent from recordings from mutant mice corresponded with the upper range of frequencies observed for fast-firing MNs in earlier slice measurements. These results suggest that, in SOD1-linked ALS mice, symptoms are a product of abnormal MN firing due at least in part to loss of neurons that fire at high frequency, associated with altered EMG patterns and hindlimb kinematics during gait.

An ALS-Associated Mutant SOD1 Rapidly Suppresses KCNT1 (Slack) Na+-Activated K+ Channels in Aplysia Neurons.

Mutations that alter levels of Slack (KCNT1) Na+-activated K+ current produce devastating effects on neuronal development and neuronal function. We now find that Slack currents are rapidly suppressed by oligomers of mutant human Cu/Zn superoxide dismutase 1 (SOD1), which are associated with motor neuron toxicity in an inherited form of amyotrophic lateral sclerosis (ALS). We recorded from bag cell neurons of Aplysia californica, a model system to study neuronal excitability. We found that injection of fluorescent wild-type SOD1 (wt SOD1YFP) or monomeric mutant G85R SOD1YFP had no effect on net ionic currents measured under voltage clamp. In contrast, outward potassium currents were significantly reduced by microinjection of mutant G85R SOD1YFP that had been preincubated at 37°C or of cross-linked dimers of G85R SOD1YFP. Reduction of potassium current was also seen with multimeric G85R SOD1YFP of ∼300 kDa or >300 kDa that had been cross-linked. In current clamp recordings, microinjection of cross-linked 300 kDa increased excitability by depolarizing the resting membrane potential, and decreasing the latency of action potentials triggered by depolarization. The effect of cross-linked 300 kDa on potassium current was reduced by removing Na+ from the bath solution, or by knocking down levels of Slack using siRNA. It was also prevented by pharmacological inhibition of ASK1 (apoptosis signal-regulating kinase 1) or of c-Jun N-terminal kinase, but not by an inhibitor of p38 mitogen-activated protein kinase. These results suggest that soluble mutant SOD1 oligomers rapidly trigger a kinase pathway that regulates the activity of Na+-activated K+ channels in neurons.SIGNIFICANCE STATEMENT Slack Na+-activated K+ channels (KCNT1, KNa1.1) regulate neuronal excitability but are also linked to cytoplasmic signaling pathways that control neuronal protein translation. Mutations that alter the amplitude of these currents have devastating effects on neuronal development and function. We find that injection of oligomers of mutant superoxide dismutase 1 (SOD1) into the cytoplasm of invertebrate neurons rapidly suppresses these Na+-activated K+ currents and that this effect is mediated by a MAP kinase cascade, including ASK1 and c-Jun N-terminal kinase. Because amyotrophic lateral sclerosis is a fatal adult-onset neurodegenerative disease produced by mutations in SOD1 that cause the enzyme to form toxic oligomers, our findings suggest that suppression of Slack channels may be an early step in the progression of the disease.

Carrying capacity in a heterogeneous environment with habitat connectivity.

A large body of theory predicts that populations diffusing in heterogeneous environments reach higher total size than if non-diffusing, and, paradoxically, higher size than in a corresponding homogeneous environment. However, this theory and its assumptions have not been rigorously tested. Here, we extended previous theory to include exploitable resources, proving qualitatively novel results, which we tested experimentally using spatially diffusing laboratory populations of yeast. Consistent with previous theory, we predicted and experimentally observed that spatial diffusion increased total equilibrium population abundance in heterogeneous environments, with the effect size depending on the relationship between r and K. Refuting previous theory, however, we discovered that homogeneously distributed resources support higher total carrying capacity than heterogeneously distributed resources, even with species diffusion. Our results provide rigorous experimental tests of new and old theory, demonstrating how the traditional notion of carrying capacity is ambiguous for populations diffusing in spatially heterogeneous environments.

Dispersal and spatial heterogeneity: single species.

A recent result for a reaction-diffusion equation is that a population diffusing at any rate in an environment in which resources vary spatially will reach a higher total equilibrium biomass than the population in an environment in which the same total resources are distributed homogeneously. This has so far been proven by Lou for the case in which the reaction term has only one parameter, m(x), varying with spatial location x, which serves as both the intrinsic growth rate coefficient and carrying capacity of the population. However, this striking result seems rather limited when applies to real populations. In order to make the model more relevant for ecologists, we consider a logistic reaction term, with two parameters, r (x) for intrinsic growth rate, and K(x) for carrying capacity. When r (x) and K(x) are proportional, the logistic equation takes a particularly simple form, and the earlier result still holds. In this paper we have established the result for the more general case of a positive correlation between r (x) and K(x) when dispersal rate is small. We review natural and laboratory systems to which these results are relevant and discuss the implications of the results to population theory and conservation ecology.

UCS proteins: chaperones for myosin and co-chaperones for Hsp90.

The UCS (UNC-45/CRO1/She4p) family of proteins has emerged as chaperones that are specific for the folding, assembly and function of myosin. These proteins participate in various important myosin-dependent cellular processes that include myofibril organization and muscle functions, cell differentiation, cardiac and skeletal muscle development, cytokinesis and endocytosis. Mutations in the genes that code for UCS proteins cause serious defects in these actomyosin-based processes. Homologs of UCS proteins can be broadly divided into (1) animal UCS proteins, generally known as UNC-45 proteins, which contain an N-terminal tetratricopeptide repeat (TPR) domain in addition to the canonical UCS domain, and (2) fungal UCS proteins, which lack the TPR domain. Structurally, except for TPR domain, both sub-classes of UCS proteins comprise of several irregular armadillo (ARM) repeats that are divided into two-domain architecture: a combined central-neck domain and a C-terminal UCS domain. Structural analyses suggest that UNC-45 proteins form elongated oligomers that serve as scaffolds to recruit Hsp90 and/or Hsp70 to form a multi-protein chaperoning complex that assists myosin heads to fold and simultaneously organize them into myofibrils. Similarly, fungal UCS proteins may dimerize to promote folding of non-muscle myosins as well as determine their step size along actin filaments. These findings confirm UCS proteins as a new class of myosin-specific chaperones and co-chaperones for Hsp90. This chapter reviews the implications of the outcome of studies on these proteins in cellular processes such as muscle formation, and disease states such as myopathies and cancer.

Molecular chaperone Hsp110 rescues a vesicle transport defect produced by an ALS-associated mutant SOD1 protein in squid axoplasm.

Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is associated with motor neuron toxicity and death in an inherited form of amyotrophic lateral sclerosis (ALS; Lou Gehrig disease). One aspect of toxicity in motor neurons involves diminished fast axonal transport, observed both in transgenic mice and, more recently, in axoplasm isolated from squid giant axons. The latter effect appears to be directly mediated by misfolded SOD1, whose addition activates phosphorylation of p38 MAPK and phosphorylation of kinesin. Here, we observe that several different oligomeric states of a fusion protein, comprising ALS-associated human G85R SOD1 joined with yellow fluorescent protein (G85R SOD1YFP), which produces ALS in transgenic mice, inhibited anterograde transport when added to squid axoplasm. Inhibition was blocked both by an apoptosis signal-regulating kinase 1 (ASK1; MAPKKK) inhibitor and by a p38 inhibitor, indicating the transport defect is mediated through the MAPK cascade. In further incubations, we observed that addition of the mammalian molecular chaperone Hsc70, abundantly associated with G85R SOD1YFP in spinal cord of transgenic mice, exerted partial correction of the transport defect, associated with diminished phosphorylation of p38. Most striking, the addition of the molecular chaperone Hsp110, in a concentration substoichiometric to the mutant SOD1 protein, completely rescued both the transport defect and the phosphorylation of p38. Hsp110 has been demonstrated to act as a nucleotide exchange factor for Hsc70 and, more recently, to be able to cooperate with it to mediate protein disaggregation. We speculate that it can cooperate with endogenous squid Hsp(c)70 to mediate binding and/or disaggregation of mutant SOD1 protein, abrogating toxicity.

The myosin-binding UCS domain but not the Hsp90-binding TPR domain of the UNC-45 chaperone is essential for function in Caenorhabditis elegans.

The UNC-45 family of molecular chaperones is expressed in metazoan organisms from Caenorhabditis elegans to humans. The UNC-45 protein is essential in C. elegans for early body-wall muscle cell development and A-band assembly. We show that the myosin-binding UCS domain of UNC-45 alone is sufficient to rescue lethal unc-45 null mutants arrested in embryonic muscle development and temperature-sensitive loss-of-function unc-45 mutants defective in worm A-band assembly. Removal of the Hsp90-binding TPR domain of UNC-45 does not affect rescue. Similar results were obtained with overexpression of the same fragments in wild-type nematodes when assayed for diminution of myosin accumulation and assembly. Titration experiments show that, on a per molecule basis, UCS has greater activity in C. elegans muscle in vivo than full-length UNC-45 protein, suggesting that UNC-45 is inhibited by either the TPR domain or its interaction with the general chaperone Hsp90. In vitro experiments with purified recombinant C. elegans Hsp90 and UNC-45 proteins show that they compete for binding to C. elegans myosin. Our in vivo genetic and in vitro biochemical experiments are consistent with a novel inhibitory role for Hsp90 with respect to UNC-45 action.

Camel nanobody-based B7-H3 CAR-T cells show high efficacy against large solid tumours.

Rational design of chimeric antigen receptor T (CAR-T) cells based on the recognition of antigenic epitopes capable of evoking the most potent CAR activation is an important objective in optimizing immune therapy. In solid tumors, the B7-H3 transmembrane protein is an emerging target that harbours two distinct epitope motifs, IgC and IgV, in its ectodomain. Here, we generate dromedary camel nanobodies targeting B7-H3 and demonstrate that CAR-T cells, based on the nanobodies recognizing the IgC but not IgV domain, had potent antitumour activity against large tumors in female mice. These CAR-T cells are characterized by highly activated T cell signaling and significant tumor infiltration. Single-cell transcriptome RNA sequencing coupled with functional T-cell proteomics analysis uncovers the top-upregulated genes that might be critical for the persistence of polyfunctional CAR-T cells in mice. Our results highlight the importance of the specific target antigen epitope in governing optimal CAR-T activity and provide a nanobody-based B7-H3 CAR-T product for use in solid tumor therapy.

The IgG4 hinge with CD28 transmembrane domain improves VHH-based CAR T cells targeting a membrane-distal epitope of GPC1 in pancreatic cancer.

Heterogeneous antigen expression is a key barrier influencing the activity of chimeric antigen receptor (CAR) T cells in solid tumors. Here, we develop CAR T cells targeting glypican-1 (GPC1), an oncofetal antigen expressed in pancreatic cancer. We report the generation of dromedary camel VHH nanobody (D4)-based CAR T cells targeting GPC1 and the optimization of the hinge (H) and transmembrane domain (TM) to improve activity. We find that a structurally rigid IgG4H and CD28TM domain brings the two D4 fragments in proximity, driving CAR dimerization and leading to enhanced T-cell signaling and tumor regression in pancreatic cancer models with low antigen density in female mice. Furthermore, single-cell-based proteomic and transcriptomic analysis of D4-IgG4H-CD28TM CAR T cells reveals specific genes (e.g., HMGB1) associated with high T-cell polyfunctionality. This study demonstrates the potential of VHH-based CAR T for pancreatic cancer therapy and provides an engineering strategy for developing potent CAR T cells targeting membrane-distal epitopes.

Multiomics and spatial mapping characterizes human CD8+ T cell states in cancer.

Clinically relevant immunological biomarkers that discriminate between diverse hypofunctional states of tumor-associated CD8+ T cells remain disputed. Using multiomics analysis of CD8+ T cell features across multiple patient cohorts and tumor types, we identified tumor niche-dependent exhausted and other types of hypofunctional CD8+ T cell states. CD8+ T cells in "supportive" niches, like melanoma or lung cancer, exhibited features of tumor reactivity-driven exhaustion (CD8+ TEX). These included a proficient effector memory phenotype, an expanded T cell receptor (TCR) repertoire linked to effector exhaustion signaling, and a cancer-relevant T cell-activating immunopeptidome composed of largely shared cancer antigens or neoantigens. In contrast, "nonsupportive" niches, like glioblastoma, were enriched for features of hypofunctionality distinct from canonical exhaustion. This included immature or insufficiently activated T cell states, high wound healing signatures, nonexpanded TCR repertoires linked to anti-inflammatory signaling, high T cell-recognizable self-epitopes, and an antiproliferative state linked to stress or prodeath responses. In situ spatial mapping of glioblastoma highlighted the prevalence of dysfunctional CD4+:CD8+ T cell interactions, whereas ex vivo single-cell secretome mapping of glioblastoma CD8+ T cells confirmed negligible effector functionality and a promyeloid, wound healing-like chemokine profile. Within immuno-oncology clinical trials, anti-programmed cell death protein 1 (PD-1) immunotherapy facilitated glioblastoma's tolerogenic disparities, whereas dendritic cell (DC) vaccines partly corrected them. Accordingly, recipients of a DC vaccine for glioblastoma had high effector memory CD8+ T cells and evidence of antigen-specific immunity. Collectively, we provide an atlas for assessing different CD8+ T cell hypofunctional states in immunogenic versus nonimmunogenic cancers.