Successful AAV8 readministration: Suppression of capsid-specific neutralizing antibodies by a combination treatment of bortezomib and CD20 mAb in a mouse model of Pompe disease.

BACKGROUND: A major challenge to adeno-associated virus (AAV)-mediated gene therapy is the presence of anti-AAV capsid neutralizing antibodies (NAbs), which can block viral vector transduction even at very low titers. In the present study, we examined the ability of a combination immunosuppression (IS) treatment with bortezomib and a mouse-specific CD20 monoclonal antibody to suppress anti-AAV NAbs and enable readministration of AAV vectors of the same capsid in mice. METHODS: An AAV8 vector (AAV8-CB-hGAA) that ubiquitously expresses human α-glucosidase was used for initial gene therapy and a second AAV8 vector (AAV8-LSP-hSEAP) that contains a liver-specific promoter to express human secreted embryonic alkaline phosphatase (hSEAP) was used for AAV readministration. Plasma samples were used for determination of anti-AAV8 NAb titers. Cells isolated from whole blood, spleen, and bone marrow were analyzed for B-cell depletion by flow cytometry. The efficiency of AAV readministration was determined by the secretion of hSEAP in blood. RESULTS: In näive mice, an 8-week IS treatment along with AAV8-CB-hGAA injection effectively depleted CD19+ B220+ B cells from blood, spleen, and bone marrow and prevented the formation of anti-AAV8 NAbs. Following administration of AAV8-LSP-hSEAP, increasing levels of hSEAP were detected in blood for up to 6 weeks, indicating successful AAV readministration. In mice pre-immunized with AAV8-CB-hGAA, comparison of IS treatment for 8, 12, 16, and 20 weeks revealed that the 16-week IS treatment demonstrated the highest plasma hSEAP level following AAV8-LSP-hSEAP readministration. CONCLUSIONS: Our data suggest that this combination treatment is an effective IS approach that will allow retreatment of patients with AAV-mediated gene therapy. A combination IS treatment with bortezomib and a mouse-specific CD20 monoclonal antibody effectively suppressed anti-AAV NAbs in naïve mice and in mice with pre-existing antibodies, allowing successful readministration of the same AAV capsid vector.

Investigating potential immune mechanisms of trilaciclib administered prior to chemotherapy in patients with metastatic triple-negative breast cancer.

PURPOSE: In a phase II trial in patients with metastatic triple-negative breast cancer (mTNBC; NCT02978716), administering trilaciclib prior to gemcitabine plus carboplatin (GCb) enhanced T-cell activation and improved overall survival versus GCb alone. The survival benefit was more pronounced in patients with higher immune-related gene expression. We assessed immune cell subsets and used molecular profiling to further elucidate effects on antitumor immunity. METHODS: Patients with mTNBC and ≤ 2 prior chemotherapy regimens for locally recurrent TNBC or mTNBC were randomized 1:1:1 to GCb on days 1 and 8, trilaciclib prior to GCb on days 1 and 8, or trilaciclib alone on days 1 and 8, and prior to GCb on days 2 and 9. Gene expression, immune cell populations, and Tumor Inflammation Signature (TIS) scores were assessed in baseline tumor samples, with flow cytometric analysis and intracellular and surface cytokine staining used to assess immune cell populations and function. RESULTS: After two cycles, the trilaciclib plus GCb group (n = 68) had fewer total T cells and significantly fewer CD8+ T cells and myeloid-derived suppressor cells compared with baseline, with enhanced T-cell effector function versus GCb alone. No significant differences were observed in patients who received GCb alone (n = 34). Of 58 patients in the trilaciclib plus GCb group with antitumor response data, 27 had an objective response. RNA sequencing revealed a trend toward higher baseline TIS scores among responders versus non­responders. CONCLUSION: The results suggest that administering trilaciclib prior to GCb may modulate the composition and response of immune cell subsets to TNBC.

Factors Associated With a Discharge Against Medical Advice From an Emergency Department in Adult Patients With Appendicitis.

BACKGROUND: Delays in care can lead to worsened outcomes with acute appendicitis. To get timely treatment, patients must consent. OBJECTIVE: To determine if there are racial and socioeconomic differences in discharge against medical advice (DAMA) rates from an emergency department after the diagnosis of acute appendicitis. METHODS: Patients were identified retrospectively from the 2019 National Emergency Department Sample. The inclusion criteria were patients 18 years of age or older with acute appendicitis. Rates were compared using chi-square or Fisher's exact test. Odds ratios were determined using multiple logistic regression. A p value of 0.05 was used to determine statistical significance. RESULTS: The overall rate of DAMA was low (0.37%). Black patients had the highest rate, and White patients had the lowest (0.72% and 0.28%, respectively, p < 0.001). When controlling for covariates, Black patients also had a higher odds ratio (OR) for DAMA (OR 1.96, 95% confidence interval [CI] 1.29-2.97). Male patients had a higher unadjusted rate (0.47% vs. 0.26% in females, p < 0.001) and were at increased risk (OR 1.78, 95% CI 1.32-2.41). Patients between 30 and 65 years old had an increased risk (OR 1.48, 95% CI 1.10-2.0). Patients with government insurance or no insurance had higher rates than private insurance (0.57% and 0.56% vs. 0.23% respectively, p < 0.001). CONCLUSION: Race, insurance status, age, and male sex were all associated with increase in DAMA. Risk stratifying patients can help to determine how to best employ mitigations strategies. Reducing DAMA may be the next area for improving reducing disparities in appendicitis care.

Trilaciclib prior to gemcitabine plus carboplatin for metastatic triple-negative breast cancer: phase III PRESERVE 2.

Triple-negative breast cancer (TNBC) is an aggressive malignancy for which cytotoxic chemotherapy remains the backbone of treatment. Trilaciclib is an intravenous cyclin-dependent kinase 4/6 inhibitor that induces transient cell cycle arrest of hematopoietic stem and progenitor cells and immune cells during chemotherapy exposure, protecting them from chemotherapy-induced damage and enhancing immune activity. Administration of trilaciclib prior to gemcitabine plus carboplatin (GCb) significantly improved overall survival (OS) compared with GCb alone in an open-label phase II trial in patients with metastatic TNBC, potentially through protection and direct activation of immune function. The randomized, double-blind, placebo-controlled, phase III PRESERVE 2 trial will evaluate the efficacy and safety of trilaciclib administered prior to GCb in patients with locally advanced unresectable or metastatic TNBC. Clinical Trial Registration: NCT04799249 (ClinicalTrials.gov).

Cellular changes in eculizumab early responders with generalized myasthenia gravis.

Eculizumab (ECU), a C5 complement inhibitor, is approved to treat acetylcholine receptor autoantibody positive generalized myasthenia gravis (AChR MG). The clinical effect of ECU relies on inhibition of the terminal complement complex; however, the effect of ECU on lymphocytes is largely unknown. We evaluated innate and adaptive immunity among AChR MG patients (N = 3) before ECU and ≥3 months later while on stable therapy, and found reduced activation markers in memory CD4+ T cell subsets, increased regulatory T cell populations, and reduced frequencies of CXCR5+HLA-DR+CCR7+ Tfh subsets and CD11b+ migratory memory B cells. We observed increases within CD8+ T cell subsets that were terminally differentiated and senescent. Our data suggest complement inhibition with ECU modulates the adaptive immunity in patients with MG, consistent with preclinical data showing changes in complement-mediated signaling by T- and antigen-presenting cells. These findings extend our understanding of ECU's mechanism of action when treating patients with MG.

Reduced plasmablast frequency is associated with seronegative myasthenia gravis.

BACKGROUND: The immunopathology of autoimmune seronegative myasthenia gravis (SN MG) is poorly understood. Our objective was to determine immune profiles associated with a diagnosis of SN MG. METHODS: We performed high-dimensional flow cytometry on blood samples from SN MG patients (N = 68), healthy controls (N = 46), and acetylcholine receptor antibody (AChR+) MG patients (N = 27). We compared 12 immune cell subsets in SN MG to controls using logistic modeling via a discovery-replication design. An exploratory analysis fit a multinomial model comparing AChR+ MG and controls to SN MG. RESULTS: An increase in CD19+ CD20- CD38hi plasmablast frequencies was associated with lower odds of being a SN MG case in both the discovery and replication analyses (discovery P-value = .0003, replication P-value = .0021). Interleukin (IL) -21 producing helper T cell frequencies were associated with a diagnosis of AChR+ MG (P = .004). CONCLUSIONS: Reduced plasmablast frequencies are strongly associated with a SN MG diagnosis and may be a useful diagnostic biomarker in the future.

Interleukin 17-producing T helper cells and interleukin 17 orchestrate autoreactive germinal center development in autoimmune BXD2 mice.

Interleukin 17 (IL-17) is a cytokine associated with inflammation, autoimmunity and defense against some bacteria. Here we show that IL-17 can promote autoimmune disease through a mechanism distinct from its proinflammatory effects. As compared with wild-type mice, autoimmune BXD2 mice express more IL-17 and show spontaneous development of germinal centers (GCs) before they increase production of pathogenic autoantibodies. We show that blocking IL-17 signaling disrupts CD4+ T cell and B cell interactions required for the formation of GCs and that mice lacking the IL-17 receptor have reduced GC B cell development and humoral responses. Production of IL-17 correlates with upregulated expression of the genes Rgs13 and Rgs16, which encode regulators of G-protein signaling, and results in suppression of the B cell chemotactic response to the chemokine CXCL12. These findings suggest a mechanism by which IL-17 drives autoimmune responses by promoting the formation of spontaneous GCs.

Rotationally resolved UV spectroscopy of the rotamers of indole-4-carboxylic acid: Evidence for charge transfer quenching.

Rotationally resolved electronic spectra of two conformational isomers of jet-cooled indole-4-carboxylic acid (I4CA) and the deuterated forms of the acid (-COOD) and amide (-ND) groups have been obtained using a UV laser/molecular beam spectrometer. The in-plane orientation of the acid group defines the two lowest energy rotamers of I4CA. The S1 ← S0 origin bands of the two rotamers and four isotopologues have been fit to asymmetric rotor Hamiltonians in both electronic states. From the best-fit parameters, the positions of the H-atoms in the principal axis frames of each conformer have been determined and serve to unambiguously identify the syn forms (i.e., COH⋯O) of the cis and trans rotamers. The experimental S0 and S1 inertial parameters, hydrogen atom positions, and transition dipole moment (TDM) orientations are compared with the results of theoretical calculations. The TDM orientation indicates that the S1 state is the 1La state in contrast to most substituted indoles. The molecular orbital properties and natural charges are investigated to better understand the 1La/1Lb state reversal and the extent of photoinduced intramolecular charge transfer that impacts the rotamer-dependent fluorescence lifetimes.

Pretransplant Desensitization with Costimulation Blockade and Proteasome Inhibitor Reduces DSA and Delays Antibody-Mediated Rejection in Highly Sensitized Nonhuman Primate Kidney Transplant Recipients.

BACKGROUND: Patients with broad HLA sensitization have poor access to donor organs, high mortality while waiting for kidney transplant, and inferior graft survival. Although desensitization strategies permit transplantation via lowering of donor-specific antibodies, the B cell-response axis from germinal center activation to plasma cell differentiation remains intact. METHODS: To investigate targeting the germinal center response and plasma cells as a desensitization strategy, we sensitized maximally MHC-mismatched rhesus pairs with two sequential skin transplants. We administered a proteasome inhibitor (carfilzomib) and costimulation blockade agent (belatacept) to six animals weekly for 1 month; four controls received no treatment. We analyzed blood, lymph node, bone marrow cells, and serum before desensitization, after desensitization, and after kidney transplantation. RESULTS: The group receiving carfilzomib and belatacept exhibited significantly reduced levels of donor-specific antibodies (P=0.05) and bone marrow plasma cells (P=0.02) compared with controls, with a trend toward reduced lymph node T follicular helper cells (P=0.06). Compared with controls, carfilzomib- and belatacept-treated animals had significantly prolonged graft survival (P=0.02), and renal biopsy at 1 month showed significantly reduced antibody-mediated rejection scores (P=0.02). However, four of five animals with long-term graft survival showed gradual rebound of donor-specific antibodies and antibody-mediated rejection. CONCLUSIONS: Desensitization using proteasome inhibition and costimulation blockade reduces bone marrow plasma cells, disorganizes germinal center responses, reduces donor-specific antibody levels, and prolongs allograft survival in highly sensitized nonhuman primates. Most animals experienced antibody-mediated rejection with humoral-response rebound, suggesting desensitization must be maintained after transplantation using ongoing suppression of the B cell response.

Daratumumab in Sensitized Kidney Transplantation: Potentials and Limitations of Experimental and Clinical Use.

BACKGROUND: Donor-specific antibodies are associated with increased risk of antibody-mediated rejection and decreased allograft survival. Therefore, reducing the risk of these antibodies remains a clinical need in transplantation. Plasma cells are a logical target of therapy given their critical role in antibody production. METHODS: To target plasma cells, we treated sensitized rhesus macaques with daratumumab (anti-CD38 mAb). Before transplant, we sensitized eight macaques with two sequential skin grafts from MHC-mismatched donors; four of them were also desensitized with daratumumab and plerixafor (anti-CXCR4). We also treated two patients with daratumumab in the context of transplant. RESULTS: The animals treated with daratumumab had significantly reduced donor-specific antibody levels compared with untreated controls (57.9% versus 13% reduction; P<0.05) and prolonged renal graft survival (28.0 days versus 5.2 days; P<0.01). However, the reduction in donor-specific antibodies was not maintained because all recipients demonstrated rapid rebound of antibodies, with profound T cell-mediated rejection. In the two clinical patients, a combined heart and kidney transplant recipient with refractory antibody-mediated rejection and a highly sensitized heart transplant candidate, we also observed a significant decrease in class 1 and 2 donor-specific antibodies that led to clinical improvement of antibody-mediated rejection and to heart graft access. CONCLUSIONS: Targeting CD38 with daratumumab significantly reduced anti-HLA antibodies and anti-HLA donor-specific antibodies in a nonhuman primate model and in two transplant clinical cases before and after transplant. This supports investigation of daratumumab as a potential therapeutic strategy; however, further research is needed regarding its use for both antibody-mediated rejection and desensitization.

Point Cloud Scene Completion of Obstructed Building Facades with Generative Adversarial Inpainting.

Collecting 3D point cloud data of buildings is important for many applications such as urban mapping, renovation, preservation, and energy simulation. However, laser-scanned point clouds are often difficult to analyze, visualize, and interpret due to incompletely scanned building facades caused by numerous sources of defects such as noise, occlusions, and moving objects. Several point cloud scene completion algorithms have been proposed in the literature, but they have been mostly applied to individual objects or small-scale indoor environments and not on large-scale scans of building facades. This paper introduces a method of performing point cloud scene completion of building facades using orthographic projection and generative adversarial inpainting methods. The point cloud is first converted into the 2D structured representation of depth and color images using an orthographic projection approach. Then, a data-driven 2D inpainting approach is used to predict the complete version of the scene, given the incomplete scene in the image domain. The 2D inpainting process is fully automated and uses a customized generative-adversarial network based on Pix2Pix that is trainable end-to-end. The inpainted 2D image is finally converted back into a 3D point cloud using depth remapping. The proposed method is compared against several baseline methods, including geometric methods such as Poisson reconstruction and hole-filling, as well as learning-based methods such as the point completion network (PCN) and TopNet. Performance evaluation is carried out based on the task of reconstructing real-world building facades from partial laser-scanned point clouds. Experimental results using the performance metrics of voxel precision, voxel recall, position error, and color error showed that the proposed method has the best performance overall.

B cells in the pathophysiology of myasthenia gravis.

Myasthenia gravis (MG) is an archetypal autoimmune disease. The pathology is characterized by autoantibodies to the acetylcholine receptor (AChR) in most patients or to muscle-specific tyrosine kinase (MuSK) in others and to a growing number of other postsynaptic proteins in smaller subsets. A decrease in the number of functional AChRs or functional interruption of the AChR within the muscle end plate of the neuromuscular junction is caused by pathogenic autoantibodies. Although the molecular immunology underpinning the pathology is well understood, much remains to be learned about the cellular immunology contributing to the production of autoantibodies. This Review documents research concerning the immunopathology of MG, bringing together evidence principally from human studies with an emphasis on the role of adaptive immunity and B cells in particular. Proposed mechanisms for autoimmunity, which take into account that different types of MG may incorporate divergent immunopathology, are offered. Muscle Nerve 57: 172-184, 2018.

Randomized controlled trial of postoperative belladonna and opium rectal suppositories in vaginal surgery.

BACKGROUND: After vaginal surgery, oral and parenteral narcotics are used commonly for pain relief, and their use may exacerbate the incidence of sedation, nausea, and vomiting, which ultimately delays convalescence. Previous studies have demonstrated that rectal analgesia after surgery results in lower pain scores and less intravenous morphine consumption. Belladonna and opium rectal suppositories may be used to relieve pain and minimize side effects; however, their efficacy has not been confirmed. OBJECTIVE: We aimed to evaluate the use of belladonna and opium suppositories for pain reduction in vaginal surgery. MATERIALS AND METHODS: A prospective, randomized, double-blind, placebo-controlled trial that used belladonna and opium suppositories after inpatient or outpatient vaginal surgery was conducted. Vaginal surgery was defined as (1) vaginal hysterectomy with uterosacral ligament suspension or (2) posthysterectomy prolapse repair that included uterosacral ligament suspension and/or colporrhaphy. Belladonna and opium 16A (16.2/60 mg) or placebo suppositories were administered rectally immediately after surgery and every 8 hours for a total of 3 doses. Patient-reported pain data were collected with the use of a visual analog scale (at 2, 4, 12, and 20 hours postoperatively. Opiate use was measured and converted into parenteral morphine equivalents. The primary outcome was pain, and secondary outcomes included pain medication, antiemetic medication, and a quality of recovery questionnaire. Adverse effects were surveyed at 24 hours and 7 days. Concomitant procedures for urinary incontinence or pelvic organ prolapse did not preclude enrollment. RESULTS: Ninety women were randomly assigned consecutively at a single institution under the care of a fellowship-trained surgeon group. Demographics did not differ among the groups with mean age of 55 years, procedure time of 97 minutes, and prolapse at 51%. Postoperative pain scores were equivalent among both groups at each time interval. The belladonna and opium group used a mean of 57 mg morphine compared with 66 mg for placebo (P=.43) in 24 hours. Patient satisfaction with recovery was similar (P=.59). Antiemetic and ketorolac use were comparable among groups. Subgroup analyses of patients with prolapse and patients <50 years old did not reveal differences in pain scores. The use of belladonna and opium suppositories was uncomplicated, and adverse effects, which included constipation and urinary retention, were similar among groups. CONCLUSION: Belladonna and opium suppositories are safe for use after vaginal surgery. Belladonna and opium suppositories did not reveal lower pain or substantially lower narcotic use. Further investigation may be warranted to identify a population that may benefit optimally from belladonna and opium use.

Rotationally resolved electronic spectroscopy of the rotamers of 1,3-dimethoxybenzene.

Conformational assignments in molecular beam experiments are often based on relative energies, although there are many other relevant parameters, such as conformer-dependent oscillator strengths, Franck-Condon factors, quantum yields and vibronic couplings. In the present contribution, we investigate the conformational landscape of 1,3-dimethoxybenzene using a combination of rotationally resolved electronic spectroscopy and high level ab initio calculations. The electronic origin of one of the three possible planar rotamers (rotamer (0,180) with both substituents pointing at each other) has not been found. Based on the calculated potential energy surface of 1,3-dimethoxybenzene in the electronic ground and lowest excited state, we show that this can be explained by a distorted non-planar geometry of rotamer (0,180) in the S1 state.

Polyfunctional T-Cell Signatures to Predict Protection from Cytomegalovirus after Lung Transplantation.

RATIONALE: Cytomegalovirus (CMV), which is one of the most common infections after lung transplantation, is associated with chronic lung allograft dysfunction and worse post-transplantation survival. Current approaches for at-risk patients include a fixed duration of antiviral prophylaxis despite the associated cost and side effects. OBJECTIVES: We sought to identify a specific immunologic signature that predicted protection from subsequent CMV. METHODS: CMV-seropositive lung transplantation recipients were included in the discovery (n = 43) and validation (n = 28) cohorts. Polyfunctional CMV-specific immunity was assessed by stimulating peripheral blood mononuclear cells with CMV pp65 or IE-1 peptide pools and then by measuring T-cell expression of CD107a, IFN-γ, tumor necrosis factor-α (TNF-α), and IL-2. Recipients were prospectively monitored for subsequent viremia. A Cox proportional hazards regression model that considered cytokine responses individually and in combination was used to create a predictive model for protection from CMV reactivation. This model was then applied to the validation cohort. MEASUREMENTS AND MAIN RESULTS: Using the discovery cohort, we identified a specific combination of polyfunctional T-cell subsets to pp65 that predicted protection from subsequent CMV viremia (concordance index 0.88 [SE, 0.087]). The model included both protective (CD107a(-)/IFN-γ(+)/IL-2(+)/TNF-α(+) CD4(+) T cells, CD107a(-)/IFN-γ(+)/IL-2(+)/TNF-α(+) CD8(+) T cells) and detrimental (CD107a(+)/IFN-γ(+)/IL-2(-)/TNF-α(-) CD8(+) T cells) subsets. The model was robust in the validation cohort (concordance index 0.81 [SE, 0.103]). CONCLUSIONS: We identified and validated a specific T-cell polyfunctional response to CMV antigen stimulation that provides a clinically useful prediction of subsequent cytomegalovirus risk. This novel diagnostic approach could inform the optimal duration of individual prophylaxis.

Adaptive immune response to therapy in hmgcr autoantibody myopathy.

INTRODUCTION: We evaluated the response to immunosuppression in a case of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR)-autoantibody myopathy. METHODS: T- and B-cell subsets were determined by flow cytometry pre- and posttherapy. RESULTS: Baseline immune profiling demonstrated strikingly elevated T-follicular helper (Tfh) cells and plasmablasts. Immunosuppression resulted in clinical improvement and decreased Tfh cells, plasmablasts, and autoantibodies. CONCLUSIONS: Immune profiling in HMGCR-autoantibody myopathy suggests a B-cell-mediated disease. Tfh cells and plasmablasts may be therapeutic biomarkers.

Electronic spectra of 2- and 3-tolunitrile in the gas phase. I. A study of methyl group internal rotation via rovibronically resolved spectroscopy.

Rotationally resolved fluorescence excitation spectra of the origin bands in the S1 ← S0 transition of 2-tolunitrile (2TN) and 3-tolunitrile (3TN) have been recorded in the collision-free environment of a molecular beam. Analyses of these data provide the rotational constants of each molecule and the potential energy curves governing the internal rotation of the attached methyl groups in both electronic states. 2TN exhibits much larger barriers along this coordinate than 3TN. Interestingly, the electronic transition dipole moment in both molecules is markedly influenced by the position of the attached methyl group rather than the position of the cyano group; possible reasons for this intriguing behavior are discussed.

Intramolecular structure and dynamics of mequinol and guaiacol in the gas phase: Rotationally resolved electronic spectra of their S1 states.

The molecular structures of guaiacol (2-methoxyphenol) and mequinol (4-methoxyphenol) have been studied using high resolution electronic spectroscopy in a molecular beam and contrasted with ab initio computations. Mequinol exhibits two low frequency bands that have been assigned to electronic origins of two possible conformers of the molecule, trans and cis. Guaiacol also shows low frequency bands, but in this case, the bands have been assigned to the electronic origin and vibrational modes of a single conformer of the isolated molecule. A detailed study of these bands indicates that guaiacol has a vibrationally averaged planar structure in the ground state, but it is distorted along both in-plane and out-of-plane coordinates in the first electronically excited state. An intramolecular hydrogen bond involving the adjacent -OH and -OCH3 groups plays a major role in these dynamics.

Immune dysregulation in chronic inflammatory demyelinating polyneuropathy.

OBJECTIVE: Chronic inflammatory demyelinating polyneuropathy (CIDP) is an autoimmune disorder of the peripheral nerves with an incompletely understood underlying pathophysiology. This investigation focused on defining B and T cell frequencies, T cell functional capacity and innate immune system analysis in patients with CIDP. METHODS: By using multi-parameter flow cytometry, we examined the phenotype and function of PBMCs in 25 CIDP patients who were relatively clinically stable on treatment who met EFNS/PNS criteria, 21 patients with genetically confirmed hereditary neuropathy and 25 healthy controls. We also evaluated the regulatory T cell (Treg) inhibitory capacity by co-culturing Treg and effector T cells. RESULTS: Proinflammatory CD4 T cells, especially type 1 helper T cell (Th1) and CD8 T cells in patients with CIDP were found to have an enhanced capacity to produce inflammatory cytokines. There was no difference in frequency of Th17 regulatory cells in CIDP patients versus healthy controls, however, Treg function was impaired in CIDP patients. There was no remarkable difference in innate immune system measures. Within B cell subsets, transitional cell frequency was decreased in CIDP patients. INTERPRETATION: Patients with CIDP clinically stable on treatment continued to show evidence of a proinflammatory state with impaired Treg function. This potentially implies an inadequate suppression of ongoing inflammation not addressed by standard of care therapies as well as persistent activity of disease while on treatment. Targeting T cells, especially inhibiting Th1 and polyfunctional CD8 T cells or improving Treg cell function could be potential targets for future therapeutic research.

Differences in microenvironment of lung cancer and pleural effusions by single-cell RNA sequencing.

BACKGROUND: Direct comparison of tumor microenvironment of matched lung cancer biopsies and pleural effusions (PE) from the same patients is critical in understanding tumor biology but has not been performed. This is the first study to compare the lung cancer and PE microenvironment by single-cell RNA sequencing (scRNA-seq). METHODS: Matched lung cancer biopsies and PE were obtained prospectively from ten patients. We isolated CD45+ cells and performed scRNA-seq to compare the biopsies and PE. RESULTS: PE had a higher proportion of CD4+ T cells but lower proportion of CD8+ T cells (False detection rate, FDR = 0.0003) compared to biopsies. There was a higher proportion of naïve CD4+ T cells (FDR = 0.04) and naïve CD8+ T cells (FDR = 0.0008) in PE vs. biopsies. On the other hand, there was a higher proportion of Tregs (FDR = 0.04), effector CD8+ (FDR = 0.006), and exhausted CD8+ T cells (FDR = 0.01) in biopsies. The expression of inflammatory genes in T cells was increased in biopsies vs. PE, including TNF, IFN-É£, IL-1R1, IL-1R2, IL-2, IL-12RB2, IL-18R1, and IL-18RAP (FDR = 0.009, 0.013, 0.029, 0.043, 0.009, 0.013, 0.004, and 0.003, respectively). The gene expression of exhaustion markers in T cells was also increased in tumor biopsies including PDCD1, CTLA4, LAG 3, HAVCR2, TIGIT, and CD160 (FDR = 0.008, 0.003, 0.002, 0.011, 0.006, and 0.049, respectively). CONCLUSIONS: There is a higher proportion of naïve T cells and lower proportion of exhausted T cells and Tregs in PE compared to lung cancer biopsies, which can be leveraged for prognostic and therapeutic applications.