scholarly journals Atorvastatin Restored the CD4+ T Cell Homeostasis By Regulating the Axis of Effector T Cells and Regulatory T Cells in Immune Thrombocytopenia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 132-132
Author(s):  
Pengcheng Xu ◽  
Yajing Zhao ◽  
Ming Hou ◽  
Panpan Han
Keyword(s):  
T Cells ◽  
Lymph Nodes ◽  
Immune Tolerance ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Effector T Cells ◽  
Small Gtpase ◽  
Control Group ◽  
Coa Reductase

Abstract Immune thrombocytopenia (ITP) is an acquired autoimmune bleeding disorder, accounting for about 1/3 of clinical hemorrhagic diseases. Loss of peripheral immune tolerance through simultaneous decrease of CD4+CD25+Foxp3+ regulatory T cells (Tregs) as well as unrestricted proliferation and activation of peripheral CD4+ effector T cells underpin the pathophysiology of ITP. Atorvastatin (AT), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, could competitively combine with HMG-CoA reductase and inhibit the production of cholesterol, accompanying with the decrease of some intermediate metabolites, such as small GTPase. Recent studies have found that statins could regulate the homeostasis of effector T cells and Tregs in some autoimmune diseases and enhance bone marrow endothelial cell function in corticosteroid-resistant ITP. However, whether AT could target the Tregs/effector T cell-axis to restore the peripheral immune tolerance in ITP is unknown. To assess the effect of AT in ITP, CD4+ T cells were isolated magnetically from peripheral blood mononuclear cells of ITP patients and cultured with different doses of AT (0μM, 5μM, 10μM, 20μM) for 3 days. The activation of CD4+ T cells were analyzed by flow cytometry. It was shown that AT could significantly inhibit the expression of CD25 on CD4+ T cells, CD4+CD45RA+ naïve T cells and CD4+CD45RO+ memory T cells and impede the switching from CD45RA to RO dose-dependently. Moreover, AT was also effective in reducing the early activation of CD4+ T cells by decreasing the expression of CD69. The dampened activation of CD4+ T cells could be reversed after blocking AT by L-mevalonate (L-MA). These results suggested that AT can inhibit the activation of CD4+ T cells and naïve T cells in vitro. We further analyzed the influence of AT on the proliferation, apoptosis and cell cycle progression of CD4+ T cells. The isolated CD4+ T cells were labeled with CFSE and cultured with AT for 7 days. AT was observed to significantly inhibit the proliferation of CD4+ T cells in a dose-dependent manner and found to induce the apoptosis of CD4+ T cells with the cell arrest in G1 phase. In line with the previous studies about the promotion of Tregs after AT treatment, our in vitro study showed that the ratio of CD4+CD25+Foxp3+ Tregs among CD4+CD25+ T cells were elevated after AT treatment, suggesting that AT could increase the proportion of Treg in activated CD4+ T cells. Furthermore, as it was reported that AT could target some small GTPase to exert its regulation on T cells, we tested the regulation role of AT on the activation of Rho, Rac and Ras by western blot. It was shown that the expression of Ras and Rho of CD4+ T cells was decreased after AT administration in the culture system, and further influence on activation of small GTPase will be confirmed by pull-down assays. Finally, in the in vivo study, we established the murine passive ITP models by injecting anti-CD41 antibody and divided them randomly into AT group (AT 40mg/kg/d) and control group (same dose of PBS). The platelet count were detected every other day and the expression of CD25 and Foxp3 on CD4+T in thymus, lymph nodes, spleen and peripheral blood of mice were determined after 7 days. There was no difference on the expression of CD25+ on CD4+ T cells in peripheral blood, lymph nodes, thymus and spleen between the two groups. But increased number of Tregs in the lymph nodes, peripheral blood and spleen of the AT group and decreased number of Tregs in thymus were observed compared to the control group, suggesting that AT could induce the development of peripheral Tregs and facilitate the migration of Tregs from thymus to peripheral organs in the ITP murine models. Due to the short period of the mice model, we didn't observe a significant increase in the platelet number after AT treatment. Our current results showed that AT played an important role in regulating peripheral immune tolerance by inhibiting the activation, proliferation, expansion and survival of CD4+ T cells, whereas increasing the number of Tregs with dampened GTPase activation. The regulatory role of AT was recapitulated in the ITP murine models. This novel mechanism of AT underlies the potential therapeutic strategy for ITP. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

mTOR pathway regulates the differentiation of peripheral blood Th2/Treg cell subsets in patients with pemphigus vulgaris

2021 ◽  
Author(s):  
Kuan Lai ◽  
Wenjing Zhang ◽  
Songshan Li ◽  
Zhiwen Zhang ◽  
Shuangde Xie ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Pemphigus Vulgaris ◽  
Treg Cells ◽  
Mtor Pathway ◽  
Cd4 T Cell ◽  
Cell Ratio ◽  
Loss Of Balance

Abstract Pemphigus vulgaris (PV) is a chronic and potentially life-threatening autoimmune blistering disease. Aberrant mTOR pathway activity is involved in many autoimmune diseases. This study investigated the correlation of mTOR pathway (PI3K/AKT/mTOR/p70S6K) activity with the loss of balance in T helper 2/regulatory T (Th2/Treg) cells in the peripheral blood of PV patients. CD4+ T cells were isolated from 15 PV patients and 15 healthy controls (HCs), the ratios of Th2/CD4+ T cells and Treg/CD4+ T cells, the activity of the mTOR pathway (PI3K/AKT/mTOR/p70S6K), the transcription factors and cytokines of Th2 and Treg cells were detected. Primary CD4+ T cells from PV patients were cultured under Th2- or Treg-polarizing conditions with or without rapamycin in vitro. We found that PV patients showed significantly elevated serum IL-4 when compared with HCs, and serum IL-4 level was positively correlated with the titer of anti-Dsg1/3 antibody and disease severity, while the serum TGF-β level was negatively correlated with the titer of anti-Dsg3 antibody and disease severity. Meanwhile, PV patients showed increased Th2/CD4+ T cell ratio; decreased Treg/CD4+ T cell ratio; elevated mRNA of PI3K, AKT, mTOR and protein of PI3K (P85), AKT, p-AKT (Ser473), mTOR, p-mTOR (Ser2448), p-p70S6K (Thr389), GATA3; reduced protein of forkhead box protein 3. Rapamycin inhibited Th2 cell differentiation and promoted Treg cell differentiation in vitro. These data suggest a close association between mTOR pathway activation and the loss of balance in Th2/Treg cells in peripheral blood of PV patients. Inhibiting mTORC1 can help restore the Th2/Treg balance.

565 A novel long-acting interleukin-7 agonist, NT-I7, increases cytotoxic CD8+ T cells and enhances survival in mouse glioma models

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A599-A599
Author(s):  
Subhajit Ghosh ◽  
Ran Yan ◽  
Sukrutha Thotala ◽  
Arijita Jash ◽  
Anita Mahadevan ◽  
...  
Keyword(s):  
T Cells ◽  
Lymph Nodes ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Standard Of Care ◽  
Control Group ◽  
Cervical Lymph Nodes ◽  
Cd8 Cells ◽  
Interleukin 7 ◽  
Long Acting

BackgroundRadiation (RT) and temozolomide (TMZ), which are standard of care for patients with glioblastoma (GBM), can cause prolonged severe lymphopenia. Lymphopenia, in turn, is an independent risk factor for shorter survival. Interleukin-7 (IL-7) is a cytokine that is required for T cell homeostasis and proliferation. IL-7 levels are inappropriately low in GBM patients with lymphopenia. NT-I7 (efineptakin alfa) is a long-acting recombinant human IL-7 that supports the proliferation and survival CD4+ and CD8+ cells in both human and mice. We tested whether NT-I7 rescues treatment-induced lymphopenia and improves survival.MethodsImmunocompetent C57BL/6 mice bearing two intracranial glioma models (GL261 and CT2A) were treated with RT (1.8 Gy/day x 5 days), TMZ (33 mg/kg/day x 5 days) and/or NT-I7 (10 mg/kg on the final day of RT completion). We profiled the CD3, CD8, CD4, FOXP3 cells in peripheral blood over time. We also immunoprofiled cervical lymph nodes, bone marrow, thymus, spleen, and the tumor 6 days after NT-I7 treatment. Survival was monitored daily.ResultsMedian survival in mice treated with NT-I7 combined with RT was significantly longer than RT alone (GL261: 40d vs 34d, p<0.0021; CT2A: 90d vs 40d, p<0.0499) or NT-I7 alone (GL261: 40d vs 24d, p<0.008; CT2A: 90d vs 32d, p<0.0154). NT-I7 with RT was just as effective as NT-I7 combined with RT and TMZ in both GL261(40d vs 47d) and CT2A (90d vs 90d). Cytotoxic CD8+ T cells were increased in both peripheral blood (0.66 x 105 to 3.34 x 105; P≤0.0001) and tumor (0.53 x 103 to 1.83 x 103; P≤0.0001) in mice treated with NT-I7 when compared to control. Similarly, NT-I7 in combination with RT increased the CD8+ T cells in peripheral blood (0.658 x 105 to 1.839 x 105 P≤0.0001) when compared to RT alone. There were decreases in tumor infiltrating FOXP3+ T-reg cells in mice treated with NT-I7 (1.9 x 104 to 0.75 x 104 P≤0.0001) and NT-I7 + RT (1.9 x 104 to 0.59 x 104 P≤0.0001) when compared to the control group without NT-I7. In addition, NT- I7 treatment increased CD8+ T cells in thymus, spleen, and lymph nodes.ConclusionsNT-I7 enhances cytotoxic CD8+ T lymphocytes systemically and in the tumor microenvironment, and improves survival. A phase I/II trial to evaluate NT-I7 in patients with high-grade gliomas is ongoing (NCT03687957).

scholarly journals CD73+ CD127high Long-Term Memory CD4 T Cells Are Highly Proliferative in Response to Recall Antigens and Are Early Targets in HIV-1 Infection

2021 ◽  
Vol 22 (2) ◽  
pp. 912
Author(s):  
Nabila Seddiki ◽  
John Zaunders ◽  
Chan Phetsouphanh ◽  
Vedran Brezar ◽  
Yin Xu ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Significant Proportion ◽  
Long Term Memory ◽  
Ki 67 ◽  
Memory Cd4 T Cells ◽  
Hiv 1

HIV-1 infection rapidly leads to a loss of the proliferative response of memory CD4+ T lymphocytes, when cultured with recall antigens. We report here that CD73 expression defines a subset of resting memory CD4+ T cells in peripheral blood, which highly express the α-chain of the IL-7 receptor (CD127), but not CD38 or Ki-67, yet are highly proliferative in response to mitogen and recall antigens, and to IL-7, in vitro. These cells also preferentially express CCR5 and produce IL-2. We reasoned that CD73+ memory CD4+ T cells decrease very early in HIV-1 infection. Indeed, CD73+ memory CD4+ T cells comprised a median of 7.5% (interquartile range: 4.5–10.4%) of CD4+ T cells in peripheral blood from healthy adults, but were decreased in primary HIV-1 infection to a median of 3.7% (IQR: 2.6–6.4%; p = 0.002); and in chronic HIV-1 infection to 1.9% (IQR: 1.1–3%; p < 0.0001), and were not restored by antiretroviral therapy. Moreover, we found that a significant proportion of CD73+ memory CD4+ T cells were skewed to a gut-homing phenotype, expressing integrins α4 and β7, CXCR3, CCR6, CD161 and CD26. Accordingly, 20% of CD4+ T cells present in gut biopsies were CD73+. In HIV+ subjects, purified CD73+ resting memory CD4+ T cells in PBMC were infected with HIV-1 DNA, determined by real-time PCR, to the same level as for purified CD73-negative CD4+ T cells, both in untreated and treated subjects. Therefore, the proliferative CD73+ subset of memory CD4+ T cells is disproportionately reduced in HIV-1 infection, but, unexpectedly, their IL-7 dependent long-term resting phenotype suggests that residual infected cells in this subset may contribute significantly to the very long-lived HIV proviral DNA reservoir in treated subjects.

scholarly journals In Vivo Detection of Dendritic Cell Antigen Presentation to CD4+ T Cells

1997 ◽  
Vol 185 (12) ◽  
pp. 2133-2141 ◽  
Author(s):  
Elizabeth Ingulli ◽  
Anna Mondino ◽  
Alexander Khoruts ◽  
Marc K. Jenkins
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lymph Nodes ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Delayed Type Hypersensitivity ◽  
Physical Interaction

Although lymphoid dendritic cells (DC) are thought to play an essential role in T cell activation, the initial physical interaction between antigen-bearing DC and antigen-specific T cells has never been directly observed in vivo under conditions where the specificity of the responding T cells for the relevant antigen could be unambiguously assessed. We used confocal microscopy to track the in vivo location of fluorescent dye-labeled DC and naive TCR transgenic CD4+ T cells specific for an OVA peptide–I-Ad complex after adoptive transfer into syngeneic recipients. DC that were not exposed to the OVA peptide, homed to the paracortical regions of the lymph nodes but did not interact with the OVA peptide-specific T cells. In contrast, the OVA peptide-specific T cells formed large clusters around paracortical DC that were pulsed in vitro with the OVA peptide before injection. Interactions were also observed between paracortical DC of the recipient and OVA peptide-specific T cells after administration of intact OVA. Injection of OVA peptide-pulsed DC caused the specific T cells to produce IL-2 in vivo, proliferate, and differentiate into effector cells capable of causing a delayed-type hypersensitivity reaction. Surprisingly, by 48 h after injection, OVA peptide-pulsed, but not unpulsed DC disappeared from the lymph nodes of mice that contained the transferred TCR transgenic population. These results demonstrate that antigen-bearing DC directly interact with naive antigen-specific T cells within the T cell–rich regions of lymph nodes. This interaction results in T cell activation and disappearance of the DC.

Platelet-Targeted Gene Transfer Induces Immune Tolerance through Two Distinct Pathways

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4423-4423
Author(s):  
Xiaofeng Luo ◽  
Juan Chen ◽  
Jocelyn Schroeder ◽  
Christina K Baumgartner ◽  
Jianda Hu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Lymph Nodes ◽  
Immune Tolerance ◽  
Cd4 T Cells ◽  
Specific Gene ◽  
Control Vector ◽  
Unrelated Control ◽  
Antibody Titers ◽  
Spleen And Lymph Nodes

Abstract Our previous studies using hemophilia A and B mouse models have demonstrated that targeting FVIII or FIX expression to platelets under control of the aIIb promoter through lentivirus-mediated delivery to hematopoietic stem cells (HSCs) results in transgene protein expression and storage in platelet a-granules and that platelet-derived FVIII or FIX not only restores hemostasis but also induces immune tolerance in transduced recipients. In the current studies, we explored how immune tolerance is induced after platelet-specific gene therapy and whether this approach can be applied to induce immune tolerance to a non-coagulant protein. We used ovalbumin (OVA) as a non-coagulant protein and constructed a lentiviral vector in which OVA is driven by the aIIb promoter (2bOVA). Since VWF propeptide (Vp) can reroute secreting proteins to a storage pathway, we designed another vector, 2bVpOVA, which contains Vp to secure OVA storage in platelet granules. We first confirmed that 2bOVA or 2bVpOVA lentiviral gene delivery to HSCs can induce anti-OVA immune tolerance in wild-type (WT) C57BL6 mice. 2bOVA or 2bVpOVA-transduced HSCs (CD45.2/B6) were transplanted into CD45.1/B6 recipients pre-conditioned with 6.6Gy total body irradiation (TBI). We found that 95% and 98% of OVA protein in whole blood was stored in platelets with an OVA protein level of 24.22±8.72 ng/108 platelets (n=10) and 1.41±0.73 ng/108 platelets (n=10) in 2bOVA and 2bVpOVA transduced recipients, respectively. Electronic microscope analysis demonstrated that the OVA transgene protein using both vectors was stored in transduced platelet a-granules. When the transduced recipients were immunized with OVA, anti-OVA antibody titers in both the 2bOVA group (560±68, n=10) and the 2bVpOVA group (320±34, n=10) were significantly lower than in untransduced controls (10424±2837, n=24), demonstrating that platelet-specific OVA gene delivery to HSCs can suppress the anti-OVA immune response. Of note, the titer of anti-OVA total IgG titer in 2bF8 (an unrelated control vector) transduced FVIIInull/B6 recipients without OVA immunization was 413±61 (n=12), which was not significantly different compared to the 2bOVA or 2bVpOVA group even after OVA immunization. In another unrelated control group, 2bGFP, anti-OVA titer was 84±17 (n=9), which was significantly higher than the data obtained from untransduced WT animals without immunization (33±7, n=24). Why there were various levels of anti-OVA antibody titers in unrelated vectors transduced recipients is still unclear and needed further investigation. To explore how immune suppression is established after platelet-specific gene transfer, we transduced HSCs from OVA-specific TCR transgenic (OTII/CD45.2) mice with 2bOVA, 2bVpOVA, or 2bGFP (a control vector) and transplanted into CD45.1/B6 recipients preconditioned with 6.6Gy TBI. After BM reconstitution, the engraftments among the 3 groups were similar (86.4±2.3%, 86.2±2.2%, and 87.4±2.0%, respectively), but donor-derived CD45.2+ CD4+ T cells in the 2bOVA (0.2±0.1%, n=5) and 2bVPOVA groups (0.9±0.4%, n=6) were consistently significantly lower than in the 2bGFP group (3.1±0.9%, n=6) in peripheral blood during the entire study course. Similarly, donor-derived CD45.2+ CD4+ T cells in both spleen and lymph nodes were significantly lower in the 2bOVA and the 2bVpOVA groups compared to the 2bGFP group. However, there were no differences in either percentage or total cell number of CD45.2+ CD4+ T cells in the thymus among the 3 groups, indicating that central tolerance may not play a role in platelet-targeted gene therapy. Notably, the frequency and total number of endogenous CD4 T cells were similar in the 3 groups. Annexin-V staining revealed that the percentage of apoptotic CD45.2+ CD4+ T cells in the 2bOVA and 2bVpOVA groups were significantly higher than in the 2bGFP group in both spleen and lymph nodes, but not in the thymus. The frequency of donor-derived regulatory T cells cells in the 2bOVA and 2bVpOVA groups were significantly higher than in the 2bGFP group in peripheral blood, spleen, and lymph nodes, but not in the thymus. Taken together, our studies demonstrate that platelet-specific gene therapy induces immune tolerance through peripheral antigen-specific CD4+ T cell clone deletion and regulatory T cell induction. Thus, platelet gene therapy can be a promising approach for immune tolerance induction. Disclosures Baumgartner: Novo Nordisk: Research Funding. Shi:BloodCenter of Wisconsin: Patents & Royalties: METHOD OF INDUCING IMMUNE TOLERANCE THROUGH TARGETTED GENE EXPRESSION..

Generation of Functional Regulatory T Cells By FOXP3 Gene Transfer into CD4 T Cells from Scurfy Mice and IPEX Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2526-2526 ◽  
Author(s):  
Marianne Delville ◽  
Emmanuelle Six ◽  
Florence Bellier ◽  
Nelly Sigrist ◽  
David Zemmour ◽  
...  
Keyword(s):  
T Cells ◽  
Gene Transfer ◽  
Regulatory T Cells ◽  
Immune Tolerance ◽  
Adoptive Transfer ◽  
Cd4 T Cells ◽  
Hematopoietic Stem ◽  
Foxp3 Gene ◽  
Human Pathology

Abstract IPEX (Immunodysregulation Polyendocrinopathy Enteropathy X-linked) syndrome is the prototype of primary immunodeficiency with prevailing autoimmunity. The disease is caused by mutations in the gene encoding the transcription factor forkhead box P3 (FOXP3), which leads to the loss of function of thymus-derived CD4+CD25+ regulatory T (tTreg) cells. In IPEX patients, the absence of a functional Treg cell compartment leads to the development of multiple autoimmune manifestations (including severe enteropathy, type 1 diabetes and eczema) usually in the first months or years of life. The current treatments for IPEX syndrome include immunosuppressive, hormone replacement therapies. Unfortunately, immunosuppressive treatments are usually only partially effective and their dose is often limited because of the occurrence of infectious complications and toxicity. Currently, the only curative treatment for IPEX syndrome is allogeneic hematopoietic stem cell transplantation (HSCT). The absence of an HLA-compatible donor for all patients and their poor clinical condition particularly expose them to a risk of mortality when HLA partially compatible donors are used. For all these reasons, effective alternative therapeutic approaches are urgently needed. Various preclinical studies have shown that partial donor chimerism is sufficient for complete remission meaning that a small number of functional natural Treg is sufficient to restore immune tolerance. This suggests that a gene therapy approach designed to selectively induce a Treg program in T cells by expressing FOXP3 could be a promising potential cure for IPEX. However, several issues might compromise the success of this strategy: (i) will the introduction of FOXP3 alone be sufficient to induce a stable Treg program or will it require additional transcription factors to lock the Treg function and sustain the stability of transduced cells? (ii) Targeting effector CD4+ T cells might be an issue in terms of T-cell receptor repertoire, since the TCR repertoire of nTregs is different from the one of effector CD4+ T cells, (iii) will FOXP3-transduced T cells be able to migrate to appropriate tissues to control auto-immune reactions?, (iv) infusion of nTreg prevents the appearance of some autoimmune manifestations in murine models, however the infusion was done in prophylaxis before the appearance of the symptoms. In order to address these questions, we have developed a mouse scurfy model to evaluate the functional and stability of the correction in vivo in parallel to the characterization of gene corrected human CD4 T cells from IPEX patients. Scurfy mice develop a disease very close to human pathology due to a spontaneous mutation of Foxp3 gene. We improved Scurfy mice model to improve animal production and increase the timeline of treatement. We demonstrated that FOXP3 gene transfer into murine CD4+ T cells enable the generation of potent regulatory T cells. Indeed we showed the functional suppressive properties of the generated CD4-FOXP3 cells in an optimized flow-cytometry-based in vitro suppression assay. The ability of CD4-FOXP3 to prevent Scurfy disease by adoptive transfer in the first days of life is currently under evaluation. Similarly in humans, we demonstrated that FOXP3 gene transfer into CD4+ T cells from IPEX patients enable the generation of potent regulatory T cells, as shown through the functional in vitro suppressive properties of the generated CD4IPEX-FOXP3. Moreover comparison of the transcriptional profile of these regulatory CD4IPEX-FOXP3 cells to natural Treg by RNA-seq analysis demonstrated a good repression of cytokine transcripts (IL4/5/13/CSF2, CD40L), a strong repression of IL7R, a strong induction of IL1R2, and a moderate activation of typical Treg genes (IL2RA, IKZF2, CTLA4). Therefore, the introduction of a functional copy of the FOXP3 gene into an IPEX patient's T cells may be enough to restore immune tolerance and thus avoid the complications of allogenic HSCT. We will also discuss the challenge of generating a large, homogenous and stable population of cells in vitro for adoptive transfer and whether it can ensure long-term disease correction without generating a context of generalized immunosuppression. Disclosures No relevant conflicts of interest to declare.

scholarly journals SHIP1 Inhibition As Novel Therapeutic Approach in Chronic Lymphocytic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 894-894
Author(s):  
Veronika Ecker ◽  
Martina Braun ◽  
Tanja Neumayer ◽  
Markus Muschen ◽  
Jürgen Ruland ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
B Cells ◽  
Immune Responses ◽  
Lymph Nodes ◽  
Peripheral Blood ◽  
Immune Cell ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling

Abstract Chronic lymphocytic leukemia (CLL) is one of the most common B cell malignancies in the Western world. Malignant B cells are blocked from differentiating into immunoglobulin producing-plasma cells and clonally accumulate in the spleen, lymph nodes, bone marrow and peripheral blood. CLL is characterized by immunosuppression throughout all disease stages, which is mediated by increased numbers of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Jitschin and Braun et al., Blood 2014) and direct inhibitory effects of the malignant CLL cells on T cells (Christopoulos etal., Blood 2011). Over the past decade, significant improvement in understanding the pathogenesis of CLL has highlighted the importance of active B cell receptor (BCR) signaling. This has revealed promising targeted treatment options, including the small molecule inhibitors targeting the phosphatidylinositol-3-kinase (PI3K) signaling pathway. Idelalisib and Duvelisib are under clinical investigation for CLL treatment, however, treatment-related toxicities are limiting their application and none of these approaches are curative, highlighting the importance of functional anti-tumor immune responses in CLL for prolonged treatment efficacy. Here, we are testing a novel approach that aims to selectively target CLL B cells and simultaneously restore an appropriate immune cell function. The phosphatase SH2-domain-containing inositol 5ʹ-phosphatase 1 (SHIP1) serves as negative feedback molecule and downregulates PI3K signaling in B cells by dephosphorylating the 5`phosphate of Phosphatidylinositol (3,4,5)-trisphosphate. We hypothesize that CLL cells rely on such negative regulators for optimal survival and can only tolerate a maximum signaling level. We are therefore testing whether SHIP1 inhibition induces hypersignaling and thereby CLL cell death. Furthermore, we are investigating whether SHIP1 inhibition simultanously stimulates immune responses, as it has been shown to induce expansion of murine hematopoietic and mesenchymal stem cell compartments (Brooks et al., Stem cells 2014). 3α-Aminocholestane (3AC) is a small molecule inhibitor of SHIP1 and can be used for pharmacological inhibition. First, we investigated the expression and phosphorylation levels of SHIP1 in CLL. We found SHIP1 to be expressed at various levels in CLL peripheral blood and strongly phosphorylated compared to age-matched healthy donors. Besides, SHIP1 transcription is upregulated in lymph nodes as compared to peripheral blood, which is in line with the assumption of increased BCR signaling in secondary lymphoid organs. We then set out to investigate the consequences of SHIP1 phosphatase inhibition. Similarly, to recent findings in acute lymphoblastic leukemia (Chen et al., Nature 2015), pharmacological inhibition of SHIP1 lead to rapid cell death of CLL cells. We further investigated the mode of cell death and observed specific features of apoptosis, namely caspase 3/7 activation and phosphatidylserine exposure on the outer cell membrane. This has been tested on primary CLL patient samples and T cell leukemia/lymphoma 1 (TCL1)-driven murine CLL cells and was not observed or significantly less pronounced in other lymphoma cell lines or healthy primary B cells. To confirm the specificity of the observed effects, we genetically activated AKT with a GFP reporter in the TCL1-driven mouse model in vivo and in vitro. By tracking GFP-expressing CLL cells, we observed an initial expansion followed by rapid cell death in vitro. When we induced AKT activation in vivo, GFP+ CLL cells were not detectable in the peripheral blood, total CLL count declined upon induction and we found decreased tumor burden in the secondary lymphoid organs, particularly in the lymph nodes. In addition to the direct effects on CLL cells, we sought to investigate the impact of SHIP1 inhibition on other immune cell functions. We observed that SHIP1 inhibition lowers the activity threshold of T cells: When we stimulated a reporter cell line with suboptimal doses of anti-CD3, 3AC treatment significantly enhanced the response rate. Therefore, we propose SHIP1 as a novel interesting target in CLL. In contrast to kinase inhibition and downregulation of the BCR signaling strength, phosphatase inhibition and BCR signaling overaction provides an attractive new treatment strategy for elimination of malignant CLL cells and stimulation of immune responses. Disclosures No relevant conflicts of interest to declare.

scholarly journals Immune Tolerance Developed in Platelet-Targeted FVIII Gene Therapy in Hemophilia Mice Is CD4+ T Cell-Mediated

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1071-1071
Author(s):  
Yingyu Chen ◽  
Xiaofeng Luo ◽  
Juan Chen ◽  
Jocelyn Schroeder ◽  
Christina K Baumgartner ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Immune Tolerance ◽  
Cd4 T Cells ◽  
Memory B Cells ◽  
Cd4 T Cell ◽  
Control Group

Abstract Immune response to factor VIII (FVIII) is not only a severe complication in protein replacement therapy, but also a major concern in gene therapy of hemophilia A. Our previous studies have demonstrated that platelet-targeted FVIII (2bF8) gene therapy together with in vivo drug-selection of transduced cells can not only rescue the bleeding diathesis but also induce anti-FVIII specific immune tolerance in FVIIInull mice. In the current study, we investigated 1) whether our non-selectable lentiviral vector (LV) for the induction of platelet-FVIII expression is sufficient to induce immune tolerance and 2) which cell compartment is tolerized after platelet gene therapy. Platelet-specific FVIII expression was introduced by 2bF8LV-transduction of hematopoietic stem cells followed by syngeneic transplantation into FVIIInull mice preconditioned with 660 cGy total body irradiation (TBI) or Busulfan (Bu) plus ATG (anti-thymocyte globulin). After bone marrow transplantation and reconstitution, animals were analyzed by PCR, qPCR, FVIII:C assay, and tail clipping test to confirm the success of 2bF8 gene therapy. Sixteen weeks after transplantation, animals were challenged with recombinant human FVIII (rhF8) via retro-orbital venous administration at a dose of 50 U/kg weekly for 4 weeks. The titers of anti-FVIII inhibitory antibodies (inhibitors) were determined by Bethesda assay. The CFSE-labeled CD4 T cell proliferation assay and ELISPOT-based memory B cell maturation assay were used to determine which cell compartment is tolerized to FVIII after 2bF8 gene therapy. The level of platelet-FVIII expression was 1.44 ± 0.39 mU/108 platelets (n = 6) in the 660 cGy group, which is not significantly different from the level obtained from the Bu+ATG group [3.04 ± 1.19 mU/108 platelets (n = 6)]. Even after rhF8 challenge, no antibodies were detected in 2bF8LV-transduced recipients in either group. In contrast, all animals in the control group that did not undergo gene therapy developed various levels of inhibitors (204±97 BU/ml, n=7). The frequency of regulatory T cells in both 660 cGy TBI (11.01±0.52%) and Bu+ATG (11.02±0.68%) groups were significantly higher than the control group (8.05±0.57%). T cell proliferation assay demonstrated that CD4+ T cells from 2bF8 LV-transduced recipients that had been challenged with rhF8 did not proliferate when restimulated with rhF8 in vitro. The daughter CD4+ T cells in the group with 10 U/ml of rhF8 were 5.84 ± 2.49% (n = 6), which was not significantly different from the control group without no rhF8 stimulation (0 U/ml) (5.33 ± 1.72%). CD4+ T cells from primed FVIIInull mice did proliferate after rhF8 restimulation. The proliferated daughter cell was 13.12 ± 6.76% (n = 7) in the group with rhF8 (10 U/ml) re-stimulation, which is significantly higher than the group without rhF8 co-culture (4.99 ± 1.16%). Since FVIII-specific memory B cell maturation is CD4+ T cell dependent, we isolated CD4+ T and memory B cells from 2bF8LV-transduced or FVIIInull mice after rhF8 immunization and co-cultured with rhF8 followed by ELISPOT assay to examine the antibody secreting cells. No spots were detected when memory B cells from rhF8-primed FVIIInull mice were co-cultured with CD4+ T cells from 2bF8LV-transduced recipients. In contrast, when memory B cells from either rhF8 immunized 2bF8LV-transduced or untreated FVIIInull mice were cultured with CD4+ T cells from rhF8-primed FVIIInull mice, there were 142 and 205 anti-FVIII antibody secreting cells, respectively, detected per 106 cells seeded. These results indicate that CD4+ T cells from 2bF8LV-transduced mice are tolerized to rhF8 stimulation. In conclusion, 2bF8 lentiviral gene transfer without in vivo selection of genetically manipulated cells can introduce FVIII-specific immune tolerance in hemophilia A mice and this immune tolerance is CD4+ T cell-mediated. Disclosures Baumgartner: Novo Nordisk: Research Funding. Shi:BloodCenter of Wisconsin: Patents & Royalties: METHOD OF INDUCING IMMUNE TOLERANCE THROUGH TARGETTED GENE EXPRESSION..

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