PD-1 Inhibitory Receptor Downregulates Asparaginyl Endopeptidase and Maintains Foxp3 Transcription Factor Stability in Induced Regulatory T Cells.

CD4+ T cell differentiation into multiple T helper (Th) cell lineages is critical for optimal adaptive immune responses. This report identifies an intrinsic mechanism by which programmed death-1 receptor (PD-1) signaling imparted regulatory phenotype to Foxp3+ Th1 cells (denoted as Tbet+iTregPDL1 cells) and inducible regulatory T (iTreg) cells. Tbet+iTregPDL1 cells prevented inflammation in murine models of experimental colitis and experimental graft versus host disease (GvHD). Programmed death ligand-1 (PDL-1) binding to PD-1 imparted regulatory function to Tbet+iTregPDL1 cells and iTreg cells by specifically downregulating endo-lysosomal protease asparaginyl endopeptidase (AEP). AEP regulated Foxp3 stability and blocking AEP imparted regulatory function in Tbet+iTreg cells. Also, Aep-/- iTreg cells significantly inhibited GvHD and maintained Foxp3 expression. PD-1-mediated Foxp3 maintenance in Tbet+ Th1 cells occurred both in tumor infiltrating lymphocytes (TILs) and during chronic viral infection. Collectively, this report has identified an intrinsic function for PD-1 in maintaining Foxp3 through proteolytic pathway.

Cutting Edge: Inhibition of the Interaction of NK Inhibitory Receptors with MHC Class I Augments Antiviral and Antitumor Immunity.

NK cells recognize MHC class I (MHC-I) Ags via stochastically expressed MHC-I-specific inhibitory receptors that prevent NK cell activation via cytoplasmic ITIM. We have identified a pan anti-MHC-I mAb that blocks NK cell inhibitory receptor binding at a site distinct from the TCR binding site. Treatment of unmanipulated mice with this mAb disrupted immune homeostasis, markedly activated NK and memory phenotype T cells, enhanced immune responses against transplanted tumors, and augmented responses to acute and chronic viral infection. mAbs of this type represent novel checkpoint inhibitors in tumor immunity, potent tools for the eradication of chronic infection, and may function as adjuvants for the augmentation of the immune response to weak vaccines.

Type I interferon signaling attenuates regulatory T cell function in viral infection and in the tumor microenvironment.

Regulatory T cells (Tregs) play a cardinal role in the immune system by suppressing detrimental autoimmune responses, but their role in acute, chronic infectious diseases and tumor microenvironment remains unclear. We recently demonstrated that IFN-α/ß receptor (IFNAR) signaling promotes Treg function in autoimmunity. Here we dissected the functional role of IFNAR-signaling in Tregs using Treg-specific IFNAR deficient (IFNARfl/flxFoxp3YFP-Cre) mice in acute LCMV Armstrong, chronic Clone-13 viral infection, and in tumor models. In both viral infection and tumor models, IFNARfl/flxFoxp3YFP-Cre mice Tregs expressed enhanced Treg associated activation antigens. LCMV-specific CD8+ T cells and tumor infiltrating lymphocytes from IFNARfl/flxFoxp3YFP-Cre mice produced less antiviral and antitumor IFN-γ and TNF-α. In chronic viral model, the numbers of antiviral effector and memory CD8+ T cells were decreased in IFNARfl/flxFoxp3YFP-Cre mice and the effector CD4+ and CD8+ T cells exhibited a phenotype compatible with enhanced exhaustion. IFNARfl/flxFoxp3YFP-Cre mice cleared Armstrong infection normally, but had higher viral titers in sera, kidneys and lungs during chronic infection, and higher tumor burden than the WT controls. The enhanced activated phenotype was evident through transcriptome analysis of IFNARfl/flxFoxp3YFP-Cre mice Tregs during infection demonstrated differential expression of a unique gene signature characterized by elevated levels of genes involved in suppression and decreased levels of genes mediating apoptosis. Thus, IFN signaling in Tregs is beneficial to host resulting in a more effective antiviral response and augmented antitumor immunity.

Epitope Mapping of SERCA2a Identifies an Antigenic Determinant That Induces Mainly Atrial Myocarditis in A/J Mice.

Sarcoplasmic/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA)2a, a critical regulator of calcium homeostasis, is known to be decreased in heart failure. Patients with myocarditis or dilated cardiomyopathy develop autoantibodies to SERCA2a suggesting that they may have pathogenetic significance. In this report, we describe epitope mapping analysis of SERCA2a in A/J mice that leads us to make five observations: 1) SERCA2a contains multiple T cell epitopes that induce varying degrees of myocarditis. One epitope, SERCA2a 971-990, induces widespread atrial inflammation without affecting noncardiac tissues; the cardiac abnormalities could be noninvasively captured by echocardiography, electrocardiography, and magnetic resonance microscopy imaging. 2) SERCA2a 971-990-induced disease was associated with the induction of CD4 T cell responses and the epitope preferentially binds MHC class II/IAk rather than IEk By creating IAk/and IEk/SERCA2a 971-990 dextramers, the T cell responses were determined by flow cytometry to be Ag specific. 3) SERCA2a 971-990-sensitized T cells produce both Th1 and Th17 cytokines. 4) Animals immunized with SERCA2a 971-990 showed Ag-specific Abs with enhanced production of IgG2a and IgG2b isotypes, suggesting that SERCA2a 971-990 can potentially act as a common epitope for both T cells and B cells. 5) Finally, SERCA2a 971-990-sensitized T cells were able to transfer disease to naive recipients. Together, these data indicate that SERCA2a is a critical autoantigen in the mediation of atrial inflammation in mice and that our model may be helpful to study the inflammatory events that underlie the development of conditions such as atrial fibrillation in humans.

Selective deletion of Eos (Ikzf4) in T-regulatory cells leads to loss of suppressive function and development of systemic autoimmunity.

Eos (lkzf4) is a member of the Ikaros family of transcription factors and is preferentially expressed in T-regulatory (Treg) cells. However, the role of Eos in Treg function is controversial. One study using siRNA knock down of Eos demonstrated that it was critical for Treg suppressor function. In contrast, Treg from mice with a global deficiency of Eos had normal Treg function in vitro and in vivo. To further dissect the function of Eos in Tregs, we generated mice with a conditional knock out of Eos in Treg cells (lkzf4fl/fl X Foxp3YFP-cre, Eos cKO). Deletion of Eos in Treg resulted in activation of CD4+Foxp3- and CD8+ T cells at the age of 3 months, cellular infiltration in non-lymphoid tissues, hyperglobulinemia, and anti-nuclear antibodies. While Tregs from Eos cKO mice displayed normal suppressive function in vitro, Eos cKO mice developed severe Experimental Autoimmune Encephalomyletis (EAE) following immunization with myelin oligodendrocyte glycoprotein (MOG) and Eos cKO Treg were unable to suppress Inflammatory Bowel Disease (IBD). Eos cKO mice had decreased growth of the transplantable murine adenocarcinoma MC38 tumor accompanied by enhanced IFN-γ/TNF-α production by CD8+ T cells in tumor draining lymph nodes. Mice with a global deficiency of Eos or a deficiency of Eos only in T cells developed autoimmunity at a much older age (12 months or 7-8 months, respectively). Taken together, Eos appears to play an essential role in multiple aspects of Treg suppressor function, but also plays an as yet unknown role in the function of CD4+Foxp3- and CD8+ T cells and potentially in non-T cells.

Identification of an Epitope from Adenine Nucleotide Translocator 1 That Induces Inflammation in Heart in A/J Mice.

Heart failure, a leading cause of death in humans, can emanate from myocarditis. Although most individuals with myocarditis recover spontaneously, some develop chronic dilated cardiomyopathy. Myocarditis may result from both infectious and noninfectious causes, including autoimmune responses to cardiac antigens. In support of this notion, intracellular cardiac antigens, like cardiac myosin heavy chain-α, cardiac troponin-I, and adenine nucleotide translocator 1 (ANT1), have been identified as autoantigens in cardiac autoimmunity. Herein, we demonstrate that ANT1 can induce autoimmune myocarditis in A/J mice by generating autoreactive T cells. We show that ANT1 encompasses multiple immunodominant epitopes (namely, ANT1 21-40, ANT1 31-50, ANT1 171-190, and ANT1 181-200). Although all four peptides induce comparable T-cell responses, only ANT1 21-40 was found to be a major myocarditogenic epitope in immunized animals. The myocarditis-inducing ability of ANT1 21-40 was associated with the generation of T cells producing predominantly IL-17A, and the antigen-sensitized T cells could transfer the disease to naïve recipients. These data indicate that cardiac mitochondrial proteins can be target autoantigens in myocarditis, supporting the notion that the antigens released as a result of primary damage may contribute to the persistence of chronic inflammation through autoimmunity.

A Urinary Metabolic Signature for Multiple Sclerosis and Neuromyelitis Optica.

Urine is a metabolite-rich biofluid that reflects the body's effort to maintain chemical and osmotic homeostasis. Clinical diagnosis routinely relies on urine samples because the collection process is easy and noninvasive. Despite these advantages, urine is an under-investigated source of biomarkers for multiple sclerosis (MS). Nuclear magnetic resonance spectroscopy (NMR) has become a common approach for analyzing urinary metabolites for disease diagnosis and biomarker discovery. For illustration of the potential of urinary metabolites for diagnosing and treating MS patients, and for differentiating between MS and other illnesses, 38 urine samples were collected from healthy controls, MS patients, and neuromyelitis optica-spectrum disorder (NMO-SD) patients and analyzed with NMR, multivariate statistics, one-way ANOVA, and univariate statistics. Urine from MS patients exhibited a statistically distinct metabolic signature from healthy and NMO-SD controls. A total of 27 metabolites were differentially altered in the urine from MS and NMO-SD patients and were associated with synthesis and degradation of ketone bodies, amino acids, propionate and pyruvate metabolism, tricarboxylic acid cycle, and glycolysis. Metabolites altered in urine from MS patients were shown to be related to known pathogenic processes relevant to MS, including alterations in energy and fatty acid metabolism, mitochondrial activity, and the gut microbiota.

Low dose post-transplant cyclophosphamide and sirolimus induce mixed chimerism with CTLA4-Ig or lymphocyte depletion in an MHC-mismatched murine allotransplantation model.

Allogeneic hematopoietic cell transplantation (allo-HCT) offers a curative option for patients with certain non-malignant hematological diseases. High-dose post-transplant cyclophosphamide (PT-Cy) (200 mg/kg) and sirolimus (3 mg/kg), (HiC) synergistically induce stable mixed chimerism. Further, sirolimus and cytotoxic T lymphocyte-associated antigen-4 immunoglobulin (CTLA4-Ig), also known as Abatacept (Aba), promote immune tolerance and allograft survival. Here, in a major histocompatibility complex (MHC)-mismatched allo-HCT murine model, we combined Aba and/or T-cell depleting anti-Thy1.2 (Thy) with a lower dose of PT-Cy (50 mg/kg) and Sirolimus (3 mg/kg), (LoC). While mice in the LoC group showed graft rejection, the addition of Thy to LoC induced similar donor chimerism levels when compared to the HiC group. However, the addition of Aba to LoC led to graft acceptance only in younger mice. When Thy was added to the LoC+Aba setting, graft acceptance was restored in both age groups. Engrafted groups displayed significantly reduced frequencies of recipient-specific interferon-γ-producing T cells as well as an increased frequency in regulatory T cells (Tregs) except in the LoC+Aba group. Splenocytes from engrafted mice showed no proliferation upon restimulation with Balb/c stimulators. Collectively, in combination with Aba or Thy, LoC may be considered to reduce graft rejection in patients who undergo allo-HCT.

Co-expression of Foxp3 and Helios facilitates the identification of human T regulatory cells in health and disease.

Foxp3 is regarded as the major transcription factor for T regulatory (Treg) cells and expression of Foxp3 is used to identify and quantitate Treg cells in mouse models. However, several studies have demonstrated that human CD4+ T conventional (Tconv) cells activated in vitro by T cell receptor (TCR) stimulation can express Foxp3. This observation has raised doubt as to the suitability of Foxp3 as a Treg marker in man. Helios, a member of the Ikaros gene family, has been shown to be expressed by 80-90% of human Foxp3+ Treg cells and can potentially serve as a marker of human Treg. Here, we confirm that Foxp3 expression is readily upregulated by Tconv upon TCR stimulation in vitro, while Helios expression is not altered. More importantly, we show that Foxp3 expression is not elevated by stimulation of hTconv in a humanized mouse model of graft versus host disease (GVHD) and in patients with a wide variety of acute and chronic inflammatory diseases including sickle cell disease, acute and chronic GVHD, systemic lupus erythematosus, as well as critical COVID-19. In all patients studied, an excellent correlation was observed between the percentage of CD4+ T cells expressing Foxp3 and the percentage expressing Helios. Taken together, these studies demonstrate that Foxp3 is not induced upon Tconv cell activation in vivo and that Foxp3 expression alone can be used to quantitate Treg cells in humans. Nevertheless, the combined use of Foxp3 and Helios expression provides a more reliable approach for the characterization of Treg in humans.

Coxsackievirus B3 infection leads to the generation of cardiac myosin heavy chain-α-reactive CD4 T cells in A/J mice.

Enteroviruses like coxsackievirus B3 (CVB3) are common suspects in myocarditis/dilated cardiomyopathy patients. Autoimmunity has been proposed as an underlying mechanism, but direct evidence of its role is lacking. To delineate autoimmune response in CVB3 myocarditis, we used IA(k) dextramers for cardiac myosin heavy chain (Myhc)-α 334-352. We have demonstrated that myocarditis-susceptible A/J mice infected with CVB3 generate Myhc-α-reactive CD4 T cells and such a repertoire was absent in naïve mice as measured by proliferative response to Myhc-α 334-352 and IA(k) dextramer staining. We also detected Myhc-α 334-352 dextramer(+) cells in the hearts of CVB3-infected mice. The autoreactive T cell repertoire derived from infected mice contained a high frequency of interleukin-17-producing cells capable of inducing myocarditis in naïve recipients. The data suggest that CVB3, a bona fide pathogen of cardiovascular system that primarily infects the heart can lead to the secondary generation of autoreactive T cells and contribute to cardiac pathology.

Identification of a second mimicry epitope from Acanthamoeba castellanii that induces CNS autoimmunity by generating cross-reactive T cells for MBP 89-101 in SJL mice.

We had previously reported that Acanthamoeba castellanii (ACA) contains a mimicry epitope for proteolipid protein 139-151 capable of inducing central nervous system (CNS) autoimmunity in SJL/J mice. We now present evidence that ACA also contains a mimicry epitope for myelin basic protein (MBP) 89-101, a derivative from amoebic nicotinamide adenine dinucleotide dehydrogenase subunit 2 (NAD). The epitope, NAD 108-120, contains a discontinuous stretch of six amino acids in the core region (VVFFKNIILIGFL) sharing 46% identity with MBP 89-101 (VHFFKNIVTPRTP; identical residues are underlined). SJL mice immunized with NAD 108-120 develop encephalomyelitis similar to the disease induced by the cognate peptide. We demonstrate that NAD 108-120 induces T cells that cross-react with MBP 89-101; the antigen-sensitized T cells, which produce predominantly T helper (T(h)) 1 and T(h)17 cytokines, transfer disease in naive SJL recipients reminiscent of the disease induced with MBP 89-101. This is the first report to demonstrate that a solitary microbe can induce CNS autoimmunity by generating cross-reactive T cells for multiple myelin antigens.

Thrombospondin-1, Platelet Factor 4, and Galectin-1 Are Associated with Engraftment in Patients with Sickle Cell Disease who Underwent Haploidentical Hematopoietic Stem Cell Transplantation.

Sickle cell disease (SCD) is an inherited red blood cell disorder that leads to significant morbidity and early mortality. The most widely available curative approach remains allogeneic hematopoietic stem cell transplantation (HSCT). HLA-haploidentical (haplo) HSCT expands the donor pool considerably and is a practical alternative for these patients, but traditionally with an increased risk of allograft rejection. Biomarkers in patient plasma could potentially help predict HSCT outcome and allow treatment at an early stage to reverse or prevent graft rejection. Reliable, noninvasive methods to predict engraftment or rejection early after HSCT are needed. We sought to detect variations in the plasma proteomes of patients who engrafted compared with those who rejected their grafts. We used a mass spectrometry-based proteomics approach to identify candidate biomarkers associated with engraftment and rejection by comparing plasma samples obtained from 9 engrafted patients and 10 patients who experienced graft rejection. A total of 1378 proteins were identified, 45 of which were differentially expressed in the engrafted group compared with the rejected group. Based on bioinformatics analysis results, information from the literature, and immunoassay availability, 7 proteins-thrombospondin-1 (Tsp-1), platelet factor 4 (Pf-4), talin-1, moesin, cell division control protein 42 homolog (CDC42), galectin-1 (Gal-1), and CD9-were selected for further analysis. We compared these protein concentrations among 35 plasma samples (engrafted, n = 9; rejected, n = 10; healthy volunteers, n = 8; nontransplanted SCD, n = 8). ELISA analysis confirmed the significant up-regulation of Tsp-1, Pf-4, and Gal-1 in plasma samples from engrafted patients compared with rejected patients, healthy African American volunteers, and the nontransplanted SCD group (P < .01). By receiver operating characteristic analysis, these 3 proteins distinguished engrafted patients from the other groups (area under the curve, >0.8; P < .05). We then evaluated the concentration of these 3 proteins in samples collected pre-HSCT and at days +30, +60, +100, and +180 post-HSCT. The results demonstrate that Tsp-1 and Pf-4 stratified engrafted patients as early as day 60 post-HSCT (P < .01), and that Gal-1 was significantly higher in engrafted patients as early as day 30 post-HSCT (P < .01). We also divided the rejected group into those who experienced primary (n = 5) and secondary graft rejection (n = 5) and found that engrafted patients had significantly higher Tsp-1 levels compared with patients who developed primary graft rejection at days +60 and +100 (P < .05), as well as higher Pf-4 levels compared with patients who developed primary graft rejection at post-transplantation (PT) day 100. Furthermore, Tsp-1 levels were significantly higher at PT days 60 and 100 and Pf-4 levels were higher at PT day 100 in engrafted patients compared with those who experienced secondary graft rejection. Increased concentrations of plasma Gal-1, Tsp-1, and Pf-4 could reflect increased T regulatory cells, IL-10, and TGF-ß, which are essential players in the initiation of immunologic tolerance. These biomarkers may provide opportunities for preemptive intervention to minimize the incidence of graft rejection.

Detection of autoreactive CD4 T cells using major histocompatibility complex class II dextramers.

BACKGROUND: Tetramers are useful tools to enumerate the frequencies of antigen-specific T cells. However, unlike CD8 T cells, CD4 T cells - especially self-reactive cells - are challenging to detect with major histocompatibility complex (MHC) class II tetramers because of low frequencies and low affinities of their T cell receptors to MHC-peptide complexes. Here, we report the use of fluorescent multimers, designated MHC dextramers that contain a large number of peptide-MHC complexes per reagent. RESULTS: The utility of MHC dextramers was evaluated in three autoimmune disease models: 1) proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis in SJL/J (H-2s) mice; 2) myelin oligodendrocyte glycoprotein (MOG) 35-55-induced experimental autoimmune encephalomyelitis in C57Bl/6 (H-2b) mice; and 3) cardiac myosin heavy chain (Myhc)-α 334-352-induced experimental autoimmune myocarditis in A/J (H-2a) mice. Flow cytometrically, we demonstrate that IAs/PLP 139-151, IAb/MOG 35-55 and IAk/Myhc-α 334-352 dextramers detect the antigen-sensitized cells with specificity, and with a detection sensitivity significantly higher than that achieved with conventional tetramers. Furthermore, we show that binding of dextramers, but not tetramers, is less dependent on the activation status of cells, permitting enumeration of antigen-specific cells ex vivo. CONCLUSIONS: The data suggest that MHC dextramers are useful tools to track the generation and functionalities of self-reactive CD4 cells in various experimental systems.

Identification of novel mimicry epitopes for cardiac myosin heavy chain-α that induce autoimmune myocarditis in A/J mice.

Myocarditis is one cause of sudden cardiac death in young adolescents, and individuals affected with myocarditis can develop dilated cardiomyopathy, a frequent reason for heart transplantation. Exposure to environmental microbes has been suspected in the initiation of heart autoimmunity, but the direct causal link is lacking. We report here identification of novel mimicry epitopes that bear sequences similar to those in cardiac myosin heavy chain (MYHC)-α 334-352. These epitopes represent Bacillus spp., Magnetospirillum gryphiswaldense, Cryptococcus neoformans and Zea mays. The mimicry peptides induced varying degrees of myocarditis in A/J mice reminiscent of the disease induced with MYHC-α 334-352. We demonstrate that the mimics induce cross-reactive T cell responses for MYHC-α 334-352 as verified by MHC class II IA(k)/tetramer staining and Th-1 and Th-17 cytokines similar to those of MYHC-α 334-352. The data suggest that exposure to environmental microbes which are otherwise innocuous can predispose to heart autoimmunity by molecular mimicry.

miR-27b*, an oxidative stress-responsive microRNA modulates nuclear factor-kB pathway in RAW 264.7 cells.

Reactive oxygen species (ROS) produced in macrophages is critical for microbial killing, but they also take part in inflammation and antigen presentation functions. MicroRNAs (miRNAs) are endogenous regulators of gene expression, and they can control immune responses. To dissect the complex nature of ROS-mediated effects in macrophages, we sought to characterize miRNAs that are responsive to oxidative stress-induced with hydrogen peroxide (H(2)O(2)) in the mouse macrophage cell line, RAW 264.7. We have identified a set of unique miRNAs that are differentially expressed in response to H(2)O(2). These include miR-27a*, miR-27b*, miR-29b*, miR-24-2*, and miR-21*, all of which were downregulated except for miR-21*. By using luciferase reporter vector containing nuclear factor-kB (NF-kB) response elements, we demonstrate that overexpression of miR-27b* suppresses lipopolysaccharide-induced activation of NF-kB in RAW 264.7 cells. Our data suggest that macrophage functions can be regulated by oxidative stress-responsive miRNAs by modulating the NF-kB pathway.

TCA cycle inactivation in Staphylococcus aureus alters nitric oxide production in RAW 264.7 cells.

Inactivation of the Staphylococcus aureus tricarboxylic acid (TCA) cycle delays the resolution of cutaneous ulcers in a mouse soft tissue infection model. In this study, it was observed that cutaneous lesions in mice infected with wild-type or isogenic aconitase mutant S. aureus strains contained comparable inflammatory infiltrates, suggesting the delayed resolution was independent of the recruitment of immune cells. These observations led us to hypothesize that staphylococcal metabolism can modulate the host immune response. Using an in vitro model system involving RAW 264.7 cells, the authors observed that cells cultured with S. aureus aconitase mutant strains produced significantly lower amounts of nitric oxide (NO(•)) and an inducible nitric oxide synthase as compared to those cells exposed to wild-type bacteria. Despite the decrease in NO(•) synthesis, the expression of antigen-presentation and costimulatory molecules was similar in cells cultured with wild-type and those cultured with aconitase mutant bacteria. The data suggest that staphylococci can evade innate immune responses and potentially enhance their ability to survive in infected hosts by altering their metabolism. This may also explain the occurrence of TCA cycle mutants in clinical S. aureus isolates.

Attenuated strain of CVB3 with a mutation in the CAR-interacting region protects against both myocarditis and pancreatitis.

Coxsackievirus B3 (CVB3), is commonly implicated in myocarditis, which can lead to dilated cardiomyopathy, in addition to causing acute pancreatitis and meningitis. Yet, no vaccines are currently available to prevent this infection. Here, we describe the derivation of a live attenuated vaccine virus, termed mutant (Mt) 10, encoding a single amino acid substitution H790A within the viral protein 1, that prevents CVB3 infection in mice and protects from both myocarditis and pancreatitis in challenge studies. We noted that animals vaccinated with Mt 10 developed virus-neutralizing antibodies, predominantly containing IgG2a and IgG2b, and to a lesser extent IgG3 and IgG1. Furthermore, by using major histocompatibility complex class II dextramers and tetramers, we demonstrated that Mt 10 induces antigen-specific T cell responses that preferentially produce interferon-γ. Finally, neither vaccine recipients nor those challenged with the wild-type virus revealed evidence of autoimmunity or cardiac injury as determined by T cell response to cardiac myosin and measurement of circulating cardiac troponin I levels, respectively. Together, our data suggest that Mt 10 is a vaccine candidate that prevents CVB3 infection through the induction of neutralizing antibodies and antigen-specific T cell responses, the two critical components needed for complete protection against virus infections in vaccine studies.

Early Myeloid Derived Suppressor Cells (eMDSCs) Are Associated With High Donor Myeloid Chimerism Following Haploidentical HSCT for Sickle Cell Disease.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a widely available curative option for patients with sickle cell disease (SCD). Our original non-myeloablative haplo-HSCT trial employing post-transplant (PT) cyclophosphamide had a low incidence of GVHD but had high rejection rates. Here, we aimed to evaluate immune reconstitution following haplo-HSCT and identify cytokines and cells associated with graft rejection/engraftment. 50 cytokines and 10 immune cell subsets were screened using multiplex-ELISA and flow cytometry, respectively, at baseline and PT-Days 30, 60, 100, and 180. We observed the most significant differences in cytokine levels between the engrafted and rejected groups at PT-Day 60, corresponding with clinical findings of secondary graft rejection. Of the 44 cytokines evaluated, plasma concentrations of 19 cytokines were different between the two groups at PT-Day 60. Factor analysis suggested two independent factors. The first factor (IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, and TGFb1 contributed significantly) was strongly associated with engraftment with OR = 2.7 (95%CI of 1.4 to 5.4), whereas the second factor (GROa and IL-18 contributed significantly) was not significantly associated with engraftment. Sufficient donor myeloid chimerism (DMC) is critical for the success of HSCT; here, we evaluated immune cells among high (H) DMC (DMC≥20%) and low (L) DMC (DMC<20%) groups along with engrafted and rejected groups. We found that early myeloid-derived suppressor cell (eMDSC) frequencies were elevated in engrafted patients and patients with HDMC at PT-Day 30 (P< 0.04 & P< 0.003, respectively). 9 of 20 patients were evaluated for the source of eMDSCs. The HDMC group had high mixed chimeric eMDSCs as compared to the LDMC group (P< 0.00001). We found a positive correlation between the frequencies of eMDSCs and Tregs at PT-Day 100 (r=0.72, P <0.0007); eMDSCs at BSL and Tregs at PT-Day 100 (r=0.63, P <0.004). Of 10 immune regulatory cells and 50 cytokines, we observed mixed chimeric eMDSCs and IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, TGFb1 as potential hits which could serve as prognostic markers in predicting allograft outcome towards engraftment following haploidentical HSCT employing post-transplant cyclophosphamide. The current findings need to be replicated and further explored in a larger cohort.

Inhibiting protein phosphatase 2A increases the antitumor effect of protein arginine methyltransferase 5 inhibition in models of glioblastoma.

BACKGROUND: Despite multi-model therapy of maximal surgical resection, radiation, chemotherapy, and tumor-treating fields, the median survival of glioblastoma (GBM) patients is less than 15 months. Protein arginine methyltransferase 5 (PRMT5) catalyzes the symmetric dimethylation of arginine residues and is overexpressed in GBM. Inhibition of PRMT5 causes senescence in stem-like GBM tumor cells. LB100, a first-in-class small molecular inhibitor of protein phosphatase 2A (PP2A), can sensitize therapy-resistant tumor cells. Here, we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition. METHODS: Patient-derived primary GBM neurospheres (GBMNS), transfected with PRMT5 target-specific siRNA, were treated with LB100 and subjected to in vitro assays including PP2A activity and western blot. The intracranial mouse xenograft model was used to test the in vivo antitumor efficacy of combination treatment. RESULTS: We found that PRMT5 depletion increased PP2A activity in GBMNS. LB100 treatment significantly reduced the viability of PRMT5-depleted GBMNS compared to PRMT5-intact GBMNS. LB100 enhanced G1 cell cycle arrest induced by PRMT5 depletion. Combination therapy also increased the expression of phospho-MLKL. Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100, indicating that necroptosis caused the enhanced cytotoxicity of combination therapy. In the in vivo mouse tumor xenograft model, LB100 treatment combined with transient depletion of PRMT5 significantly decreased tumor size and prolonged survival, while LB100 treatment alone had no survival benefit. CONCLUSION: Overall, combined PRMT5 and PP2A inhibition had significantly greater antitumor effects than PRMT5 inhibition alone.