γδ T cells gain increasing attention as carriers for tumor-targeting constructs in therapeutic contexts. However, the failure to fully account for the diversity within the subset has impeded its clinical use so far. We investigated the heterogeneity of the Vγ9Vδ2 T-cell compartment by profiling the function and gene expression of single-cell clones expanded in vitro using the rapid expansion protocol (REP), which involves repeated stimulation with interleukin (IL)-2 and IL-15. Generally known to enhance the type 1 effector program in the γδ T cells, these culture conditions polarized only a proportion of the adult peripheral blood-derived clones toward “classic” type 1 effectors marked by high interferon gamma (IFN-γ) release (HIR). Unexpectedly, a substantial fraction of the clones exhibited a low-IFN-γ-releasing (LIR) profile and instead activated a type 2-like effector program, marked by IL-4 and IL-5 secretion and expression of the transcription factor GATA3. In line with this functional dichotomy, we observed coordinated transcriptional programs linking effector function to genes associated with T-cell activation, proliferation, and cytokine production. HIR clones exhibited a more activated transcriptional profile in culture compared with LIR clones. Importantly, projection of HIR and LIR gene signatures onto ex vivo single-cell transcriptomic data demonstrated that these effector states are already present in vivo as part of a continuous activation landscape within nonexpanded Vγ9Vδ2 T cells, with LIR-like states predominating in cord blood and remaining prevalent in adult peripheral blood. These findings indicate that the functional divergence observed after in vitro expansion reflects stabilization and amplification of preexisting activation states rather than culture-induced polarization. Analysis of the Vγ9Vδ2 T-cell receptor repertoire further suggested that intrinsic signaling features may modulate, but do not dictate, effector differentiation within this activation continuum. In summary, our data indicate that effector differentiation of Vγ9Vδ2 T cells is dominated by a preexisting LIR-like activation state, a finding with major implications for current γδ T-cell-based cancer immunotherapy strategies that rely on in vivo stimulation or ex vivo engineering.

Chimeric antigen receptor (CAR) T cell therapies targeting B cell maturation antigen (BCMA) are transforming treatment for relapsed or refractory multiple myeloma (RRMM). We analyze 61 RRMM patients receiving idecabtagene vicleucel (Ide-cel; n = 34) or ciltacabtagene autoleucel (Cilta-cel; n = 27) and find that Cilta-cel achieves higher complete response (CR) rates (78% vs. 38%) and longer progression-free survival. Using a longitudinal single-cell multi-omics atlas of 135 blood samples, we show that Cilta-cel induces expansion of CD4+ cytotoxic T cells associated with CR and immune-related toxicities, whereas non-CR CD8+ T cells display impaired effector programs. Among non-B cells, plasmacytoid dendritic cells (pDCs) show the highest BCMA expression and BCMA-targeted agents eradicate a blastic plasmacytoid dendritic cell neoplasm line, suggesting a novel therapeutic avenue for this disease. Greater reductions in soluble BCMA correlate with enhanced CAR T expansion and systemic inflammation. These findings reveal cellular mechanisms driving differential efficacy and toxicity of BCMA-directed immunotherapy.

Colorectal cancer (CRC) arises in the colorectal tissue driven by genetic disorder or the accumulation of somatic mutations, leading to abnormal epithelial cell growth. In this study, we employed single-nucleus multi-omics analysis, including single-nucleus RNA-seq and single-nucleus ATAC-seq, on over 100,000 high-quality nuclei to investigate the molecular landscape of both primary tissue and patient-derived organoids (PDOs). Our analysis showed that normal PDOs (N-PDOs) derived from tissue adjacent to tumors replicate the cellular composition and differentiation trajectory of colorectal crypts. In contrast, tumor PDOs (T-PDOs) showed patient-specific transcriptomic and epigenomic heterogeneity yet consistently maintained a stem cell-like state. T-PDOs retained the somatic mutation profile of the primary tumor while also exhibiting de novo mutations not detected in either the primary tumor or N-PDOs. Notably, inferred cell-cell interaction analysis highlighted the activin signaling pathway as a potential unique feature of fibroblast-epithelial interactions within the tumor microenvironment. This study provides a comprehensive view of the transition from normal to malignant colorectal epithelium and underscores the utility of PDOs as a faithful model for capturing both conserved and patient-specific features of colorectal cancer.

BACKGROUND:The effectiveness of immunotherapies against glioblastoma (GB) remains limited. A major obstacle in advancing new strategies is the reliance on non-autologous systems, which do not accurately mimic the true extent of inter-patient heterogeneity in both immune responses and tumor susceptibility. This often leads to misleading conclusions about therapeutic efficacy and targetability. METHODS:In this study, we addressed this critical gap by employing a fully autologous model. We phenotypically characterized primary αβ and γδT cells from the peripheral blood and tumors of 40 brain tumor patients, including 36 with confirmed GB, and expanded and functionally assessed the autologous anti-GB reactivity in a subset of patients. RESULTS:Notably, only Vδ2+ and Vδ2- γδT cells, but not αβT cells, recognized autologous tumors. While Vδ2- γδT cells showed activity in a subset of patients, Vδ2+ γδTILs from all patients responded to autologous GB cells in the presence of pamidronate. In patients, a higher percentage of Vδ2+ γδTILs was associated with longer overall survival. However, the potency of Vδ2+ γδTILs varied markedly between individuals, highlighting substantial inter-patient heterogeneity in γδT cell-mediated tumor recognition. This variability was driven by differences in both immune cell-intrinsic features and tumor-intrinsic factors, including expression of BTN2A1 and especially BTN3A, the ligands of the Vδ2+ γδTCR. Functional assays revealed that anti-GB reactivity was further modulated by stimulatory and inhibitory co-receptors such as NKG2D, CD94, and TIGIT. Transcriptomic analysis linked Vδ2+ γδT cell reactivity to extracellular matrix (ECM) pathways and disrupting ECM components such as LAMA5 and TGFB1 enhanced T cell responses. Knockout of ITGA3, a LAMA5 receptor, increased BTN2A1 and BTN3A expression on GB cells, improving immune recognition. CONCLUSIONS:This study demonstrates that inter-patient heterogeneity in Vδ2+ γδTIL responses to GB is driven by the extracellular matrix-BTN3A axis. Autologous systems effectively capture this heterogeneity, offering a reliable platform to identify determinants of both immune function and tumor vulnerability, insights that are essential for the rational design of γδTIL-based immunotherapies.

Glioblastoma (GB) remains the most aggressive primary brain tumor with a poor patient prognosis. Immunotherapeutic strategies are emerging as an interesting option, however the effectivity of these therapies remains low, possibly due to tumor escape mechanisms. γδT cells have been identified as interesting effector cells for immunotherapeutic strategies as they possess anti-tumor characteristics against many cancers, including GB. However, analyses characterizing activity of γδT cells against GB in a completely autologous setting are scarce. In order to investigate the influence of T cells on GB tumors in an autologous setting, we phenotypically characterized αβ and γδT cells infiltrating in the tumor and originating from peripheral blood of 36 GB patients and expanded different T cell subsets from a selection of patients (n=9) in vitro. We showed that the most prevalent T cells, αβT cells, from both tumor and peripheral blood did not show any reactivity against autologous GB tumors, while Vδ2+ and Vδ2- γδT cells were able to recognize their autologous tumor. We witnessed that γδTILs were more active than matched PBMC-derived γδT cells and highest recognition was seen for Vδ2+ TILs, however, substantial heterogeneity in potency was observed. Interestingly, Vδ2+ TILs often used multiple receptors for recognition of GB tumor cells, which varied per patient. Also, primary GB tumors showed great heterogeneity. Combined flow cytometry and transcriptomic analysis of 5 patient-derived tumors highlighted that not only different levels in BTN2A1 and BTN3A, the natural targets for Vδ2+ TCR activation, impacted recognition, but also expression of extracellular matrix related genes. One of these genes, ITGA3, was found to modulate BTN2A1 and BTN3A expression levels, suggesting a role as a novel tumor escape mechanism for preventing Vδ2+ T cell recognition.

γδT cells play a pivotal role in cancer immune surveillance, yet the current knowledge of their function across the compartments in solid tumors is meager. To address this gap, we developed a comprehensive γδT-omics platform that integrates functional screening, biomimetic migration assays, γδTCR repertoire analysis, and transcriptomic profiling. Using matched samples from 31 patients with microsatellite-stable colorectal cancer (CRC), we analyzed γδT cells from peripheral blood (PBLsγδ), adjacent colon (LPLsγδ), primary tumors (pTILsγδ), and liver metastases (mTILsγδ). This approach uncovered striking compartmentalization of γδT cell phenotypes, clonality, and function. Tumor-reactive, clonally expanded Vδ1⁺ γδT cells were enriched in primary tumors and shared transcriptional and functional features with lamina propria lymphocytes (LPLs). In contrast, Vδ1⁺ γδT cells from liver metastases lacked tumor reactivity, exhibited distinct γδTCR repertoires, and expressed transcriptional signatures associated with TGF-β-mediated suppression and cellular quiescence, suggesting they are shaped by tissue-specific environmental cues. CXCL16 secretion by tumor cells initiated Vδ1⁺ LPLsγδ migration, which was further amplified by γδTCR-mediated CCL5 induction from pTILsγδ, leading to CCR5 downregulation and subsequent entrapment of pTILsγδ within the tumor microenvironment. Accordingly, our clinical data from an independent second cohort of 69 patients showed that infiltration by pTILsγδ, but not mTILsγδ, is associated with a protective effect against CRC progression. In summary, our study offers a compartment-resolved perspective on γδT cell behavior in CRC, revealing key trafficking and functional mechanisms, and enabling the identification of novel tumor-reactive γδTCRs and migratory cues to inform immunotherapeutic strategies for both primary and metastatic CRC. ![Figure][1] ### Competing Interest Statement JK is shareholder of Gadeta. JK, ZS and DXB are inventors on patents with γδTCR related topics. JK, ZS, DXB are inventors on patents with CD277 related topics. KWF, R5911 [1]: pending:yes

Few cancers can be targeted efficiently by engineered T cell strategies. Here, we show that γδ T cell antigen receptor (γδ TCR)-mediated cancer metabolome targeting can be combined with targeting of cancer-associated stress antigens (such as NKG2D ligands or CD277) through the addition of chimeric co-receptors. This strategy overcomes suboptimal γ9δ2 TCR engagement of αβ T cells engineered to express a defined γδ TCR (TEGs) and improves serial killing, proliferation and persistence of TEGs. In vivo, the NKG2D-CD28 WT chimera enabled control only of liquid tumors, whereas the NKG2D-4-1BB CD28TM chimera prolonged persistence of TEGs and improved control of liquid and solid tumors. The CD277-targeting chimera (103-4-1BB) was the most optimal co-stimulation format, eradicating both liquid and solid tumors. Single-cell transcriptomic analysis revealed that NKG2D-4-1BB CD28TM and 103-4-1BB chimeras reprogram TEGs through NF-κB. Owing to competition with naturally expressed NKG2D in CD8 + TEGs, the NKG2D-4-1BB CD28TM chimera mainly skewed CD4 + TEGs toward adhesion, proliferation, cytotoxicity and less exhausted signatures, whereas the 103-4-1BB chimera additionally shaped the CD8 + subset toward a proliferative state.

Vγ9Vδ2T cells have the unique ability to recognize a broad range of malignant transformed cells. The tumor targeting event involving BTN2A1 and BTN3A1 dimers on the tumor cell surface is critical, leading to full activation of the TCR. Although the molecular mechanisms governing TCR engagement and T cell activation are well-characterized, the role of Vγ9Vδ2 T cells in cancer immune surveillance remains to be fully elucidated, particularly the mechanisms that enable these cells to discriminate between healthy and malignant cells at an early stage of malignant transformation. We employed two independent, genetically engineered step-wise mutagenesis models of human colorectal and breast cancer that mimic the transformation steps leading to tumor formation. We demonstrate that various single oncogenic mutations introduced into healthy organoids or cells, are sufficient to upregulate surface expressed BTN2A1 and enable Vγ9Vδ2 TCR binding to tumor cells. However, full activation of T cells through a Vγ9Vδ2TCR required additional subsequent phosphorylation of juxtamembrane (JTM) amino acids of BTN3A1, leading to the activating heterodimerization of BTN2A1 and 3A1. Using a protein interactome mapping pipeline, we identified PHLDB2, SYNJ2 and CARMIL1 as key players in controlling these delicate dual surface dynamics of BTN2A1 and 3A1 during early transformation. This mode of action allowed Vγ9Vδ2TCR T cells to control tumors in vitro and in vivo, emphasizing the crucial role of these molecules from early mutagenesis, to advanced cancer stages, and highlighting the therapeutic potential of a Vγ9Vδ2TCR. ### Competing Interest Statement Z.S., J.K., D.X.B., T.S., A.C., A.D.M., T.K. and P.D. are inventors on different patents with γδTCR sequences, recognition mechanisms and isolation strategies. J.K. is scientific advisor and shareholder of Gadeta (www.gadeta.nl).

Many countries continue to experience pertussis epidemics despite widespread vaccination. Waning protection after booster vaccination has highlighted the need for a better understanding of the immunological factors that promote durable protection. Here we apply systems vaccinology to investigate antibody responses in adolescents in the Netherlands (N = 14; NL) and the United Kingdom (N = 12; UK) receiving a tetanus-diphtheria-acellular pertussis-inactivated poliovirus (Tdap-IPV) vaccine. We report that early antiviral and interferon gene expression signatures in blood correlate to persistence of pertussis-specific antibody responses. Single-cell analyses of the innate response identified monocytes and myeloid dendritic cells (MoDC) as principal responders that upregulate antiviral gene expression and type-I interferon cytokine production. With public data, we show that Tdap vaccination stimulates significantly lower antiviral/type-I interferon responses than Tdap-IPV, suggesting that IPV may promote antiviral gene expression. Subsequent in vitro stimulation experiments demonstrate TLR-dependent, IPV-specific activation of the pro-inflammatory p38 MAP kinase pathway in MoDCs. Together, our data provide insights into the molecular host response to pertussis booster vaccination and demonstrate that IPV enhances innate immune activity associated with persistent, pertussis-specific antibody responses.

Clonal evolution of leukemia is driven by different selection pressures operating on a heterogeneous population of cells. These pressures may include therapy-induced and immune-mediated effects which eradicate the majority of cells, but which simultaneously provide a selective advantage to a minor population of cells that is genetically or transcriptionally distinct and which grow out towards relapse. We integrated single-cell RNA-seq, DNA-seq, ATAC-seq and long-read Oxford Nanopore sequencing of matched diagnosis and relapse samples of 20 pediatric AML patients with various genetic driver mutations representative of the mutational spectrum to map the genetic and transcriptional evolution of these cancers (Figure 1). We investigated whether and how differences in AML-initiating mutations impact on transcriptional programs in AML blasts and their surrounding microenvironmental cells and how the mutational and transcriptional features of these cells are impacted by cancer therapy. Specifically, we traced the progression of these traits from the point of diagnosis to the occurrence of relapse. Our single cell RNA and ATAC data showed high patient-to-patient heterogeneity, with gene expression patterns that were largely conserved between diagnosis and relapse. Analyses of the differentially expressed genes and factorization into “metaprograms” revealed gene sets that were shared between relapse samples but could not be tied to interpretable common “biological signatures”. Exome-seq revealed highly significant changes in somatic variants, most notably in members of the RAS-signaling pathway (i.e., KRAS, NRAS, PTPN11, NF1 and CBL). Interestingly, pathogenic RAS mutations were often gained, but also lost, or switched between different RAS-pathway members, indicating that RAS-mutations are associated with extensive clonal evolution/rewiring. To unveil the effect of accumulation or loss of (sub)clones with RAS mutations on the transcriptional phenotype and epigenetic landscape of leukemic blasts, we performed targeted screening of somatic variants at single-cell resolution. To this aim, we combined long-read nanopore sequencing with 10x Genomics single-cell technologies, enabling us to directly link genetic, transcriptomics and epigenetic changes in distinct (sub)clones. Taken together, we successfully reconstructed patient-specific genetic and transcriptional evolutionary trajectories in pediatric AML. This knowledge will be essential for our understanding of AML relapse and therapy resistance.

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a ‘super engager’ behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies.

We found previously that nuclear receptors (NRs) compete for heterodimerization with their common partner, retinoid X receptor (RXR), in a ligand-dependent manner. To investigate potential competition in their DNA binding, we monitored the mobility of retinoic acid receptor (RAR) and vitamin D receptor (VDR) in live cells by fluorescence correlation spec-troscopy. First, specific agonist treatment and RXR coex-pression additively increased RAR DNA binding, while both agonist and RXR were required for increased VDR DNA binding, indicating weaker DNA binding of the VDR/RXR dimer. Second, coexpression of RAR, VDR, and RXR resulted in competition for DNA binding. Without ligand, VDR reduced the DNA-bound fraction of RAR and vice versa, i.e., a fraction of RXR molecules was occupied by the competing partner. The DNA-bound fraction of either RAR or VDR was enhanced by its own and diminished by the competing NR's agonist. When treated with both ligands, the DNA-bound fraction of RAR increased as much as due to its own agonist, whereas that of VDR increased less. RXR agonist also increased DNA binding of RAR at the expense of VDR. In summary, competition be-tween RAR and VDR for RXR is also manifested in their DNA binding in an agonist-dependent manner: RAR dominates over VDR in the absence of agonist or with both agonists present. Thus, side effects of NR-ligand-based (retinoids, thiazolidine-diones) therapies may be ameliorated by other NR ligands and be at least partly explained by reduced DNA binding due to competition. Our results also complement the model of NR action by involving competition both for RXR and for DNA sites.

Celiac disease is an autoimmune disorder in which ingestion of dietary gluten triggers an immune reaction in the small intestine leading to destruction of the lining epithelium. Current treatment focusses on lifelong adherence to a gluten-free diet. Gluten-specific CD4+ T cells and cytotoxic intraepithelial CD8+ T cells have been proposed to be central in disease pathogenesis. Here we use unbiased single-cell RNA-sequencing and explore the heterogeneity of CD45+ immune cells in the human small intestine. We show altered myeloid cell transcriptomes present in active celiac lesions. CD4+ and CD8+ T cells transcriptomes show extensive changes and we define a natural intraepithelial lymphocyte population that is reduced in celiac disease. We show that the immune landscape in Celiac patients on a gluten-free diet is only partially restored compared to control samples. Altogether, we provide a single cell transcriptomic resource that can inform the immune landscape of the small intestine during Celiac disease.

Pheochromocytoma, neuroendocrine tumor, single cell RNA-sequencing, transcriptome, heterogeneity, SDHB, RET, paraganglinoma; Pheochromocytomas (PC) and paragangliomas (PG) are rare neuroendocrine tumors with varied genetic makeup and are associated with high cardiovascular morbidity and a variable risk of malignancy. The source of the transcriptional heterogeneity of the disease and the underlying biological processes that determine the outcome of PCPG remain largely unclear. We focused on PCPG tumors with germline SDHB and RET mutations, which represent distinct prognostic groups with worse or better prognoses, respectively. We applied single-nuclei RNA sequencing (snRNA-seq) to tissue samples from 11 patients and found high patient-to-patient transcriptome heterogeneity in neuroendocrine tumor cells. The tumor microenvironment also showed heterogeneous profiles, mainly contributed by macrophages of the immune cell clusters and Schwann cells of the stroma. By performing non-negative matrix factorization, we identified common transcriptional programs active in RET and SDHB, as well as distinct modules, including neuronal development, hormone synthesis and secretion, and DNA replication. Similarities between the transcriptomes of the tumor cells and those of the chromaffin- and precursor cell types suggests different developmental stages at which PC and PG tumors appear to be arrested.

Background Acute myeloid leukemia (AML) is a heterogeneous and aggressive blood cancer that results from diverse genetic aberrations in the hematopoietic stem or progenitor cells (HSPCs) leading to the expansion of blasts in the hematopoietic system. The heterogeneity and evolution of cancer blasts can render therapeutic interventions ineffective in a yet poorly understood patient-specific manner. In this study, we investigated the clonal heterogeneity of diagnosis (Dx) and relapse (Re) pairs at genetic and transcriptional levels, and unveiled the underlying pathways and genes contributing to recurrence. Methods Whole-exome sequencing was used to detect somatic mutations and large copy number variations (CNVs). Single cell RNA-seq was performed to investigate the clonal heterogeneity between Dx-Re pairs and amongst patients. Results scRNA-seq analysis revealed extensive expression differences between patients and Dx-Re pairs, even for those with the same -presumed- initiating events. Transcriptional differences between and within patients are associated with clonal composition and evolution, with the most striking differences in patients that gained large-scale copy number variations at relapse. These differences appear to have significant molecular implications, exemplified by a DNMT3A/FLT3-ITD patient where the leukemia switched from an AP-1 regulated clone at Dx to a mTOR signaling driven clone at Re. The two distinct AML1-ETO pairs share genes related to hematopoietic stem cell maintenance and cell migration suggesting that the Re leukemic stem cell-like (LSC-like) cells evolved from the Dx cells. Conclusions In summary, the single cell RNA data underpinned the tumor heterogeneity not only amongst patient blasts with similar initiating mutations but also between each Dx-Re pair. Our results suggest alternatively and currently unappreciated and unexplored mechanisms leading to therapeutic resistance and AML recurrence.

Macrophage (Mϕ) repolarization from a pro-tumor, immunosuppressive phenotype towards an anti-tumor, pro-inflammatory state represents a promising therapeutic strategy in patients with cancer1. Successful reprogramming of Mϕ in a clinical setting has not been documented. Here, we traced the evolution at single-cell resolution of a diffuse midline glioma (DMG) with H3K27M mutation which metastasized in the abdomen after placing a ventriculoperitoneal (VP) shunt and exploited this information for therapeutic decision-making. The primary tumor showed a complex cellular and genomic landscape characterized by heterogeneous cancer cells and a tumor-supportive immune microenvironment. Upon metastasis, malignant cells populated the peritoneum and triggered a massive anti-inflammatory immune cell infiltration and expansion of myeloid-derived suppressor cells (MDSCs) in peripheral blood. Hydroxychloroquine adjuvant treatment started to overcome the immunosuppressive milieu, which resulted in a decrease in peritoneal cancer cells and pro-tumor innate immune cells. Importantly, an emergence of anti-tumor, pro-inflammatory macrophages and cytotoxic T-cells was observed, accompanied by the activation of monocytes in the blood. Our study advocates the employment of single-cell technologies to better understand and inspire therapeutic regimens in patients with cancer.

SummaryCellular immunotherapies are rapidly gaining clinical importance, yet predictive platforms for modeling their mode of action are lacking. Here, we developed a dynamic immuno-organoid 3D imaging-transcriptomics platform; BEHAV3D, to unravel the behavioral and underlying molecular mechanisms of solid tumor targeting. Applied to an emerging cancer metabolome-sensing immunotherapy: TEGs, we first demonstrate targeting of multiple breast cancer subtypes. Live-tracking of over 120,000 TEGs revealed a diverse behavioral landscape and identified a ‘super engager’ cluster with serial killing capability. Inference of single-cell behavior with transcriptomics identified the gene signature of ‘super engager’ killer TEGs, which contained 27 genes with no previously described T cell function. Furthermore, guided by a dynamic type 1 interferon (IFN-I) signaling module induced by high TEG-sensitive organoids, we show that IFN-I can prime resistant organoids for TEG-mediated killing. Thus, BEHAV3D characterizes behavioral-phenotypic heterogeneity of cellular immunotherapies and holds promise for improving solid tumor-targeting in a patient-specific manner.

Single Plane Illumination Microscopy (SPIM) revolutionized time lapse imaging of live cells and organisms due to its high speed and reduced photodamage. Quantitative mapping of molecular (co)mobility by fluorescence (cross-)correlation spectroscopy (F(C)CS) in a SPIM has been introduced to reveal molecular diffusion and binding. A complementary aspect of interactions is proximity, which can be studied by Förster resonance energy transfer (FRET). Here, we extend SPIM-FCCS by alternating laser excitation, which reduces false positive cross-correlation and facilitates comapping of FRET. Thus, different aspects of interacting systems can be studied simultaneously, and molecular subpopulations can be discriminated by multiparameter analysis. After demonstrating the benefits of the method on the AP-1 transcription factor, the dimerization and DNA binding behavior of retinoic acid receptor (RAR) and retinoid X receptor (RXR) is revealed, and an extension of the molecular switch model of the nuclear receptor action is proposed. Our data imply that RAR agonist enhances RAR-RXR heterodimerization, and chromatin binding/dimerization are positively correlated. We also propose a ligand induced conformational change bringing the N-termini of RAR and RXR closer together. The RXR agonist increased homodimerization of RXR suggesting that RXR may act as an autonomous transcription factor.

Mycobacterium vaccae (NCTC 11659) is an environmental saprophytic bacterium with anti-inflammatory, immunoregulatory, and stress resilience properties. Previous studies have shown that whole, heat-killed preparations of M. vaccae prevent allergic airway inflammation in a murine model of allergic asthma. Recent studies also demonstrate that immunization with M. vaccae prevents stress-induced exaggeration of proinflammatory cytokine secretion from mesenteric lymph node cells stimulated ex vivo, prevents stress-induced exaggeration of chemically induced colitis in a model of inflammatory bowel disease, and prevents stress-induced anxiety-like defensive behavioral responses. Furthermore, immunization with M. vaccae induces anti-inflammatory responses in the brain and prevents stress-induced exaggeration of microglial priming. However, the molecular mechanisms underlying anti-inflammatory effects of M. vaccae are not known. Our objective was to identify and characterize novel anti-inflammatory molecules from M. vaccae NCTC 11659. We have purified and identified a unique anti-inflammatory triglyceride, 1,2,3-tri [Z-10-hexadecenoyl] glycerol, from M. vaccae and evaluated its effects in freshly isolated murine peritoneal macrophages. The free fatty acid form of 1,2,3-tri [Z-10-hexadecenoyl] glycerol, 10(Z)-hexadecenoic acid, decreased lipopolysaccharide-stimulated secretion of the proinflammatory cytokine IL-6 ex vivo. Meanwhile, next-generation RNA sequencing revealed that pretreatment with 10(Z)-hexadecenoic acid upregulated genes associated with peroxisome proliferator-activated receptor alpha (PPARα) signaling in lipopolysaccharide-stimulated macrophages, in association with a broad transcriptional repression of inflammatory markers. We confirmed using luciferase-based transfection assays that 10(Z)-hexadecenoic acid activated PPARα signaling, but not PPARγ, PPARδ, or retinoic acid receptor (RAR) α signaling. The effects of 10(Z)-hexadecenoic acid on lipopolysaccharide-stimulated secretion of IL-6 were prevented by PPARα antagonists and absent in PPARα-deficient mice. Future studies should evaluate the effects of 10(Z)-hexadecenoic acid on stress-induced exaggeration of peripheral inflammatory signaling, central neuroinflammatory signaling, and anxiety- and fear-related defensive behavioral responses.