scholarly journals Vitronectin-derived bioactive peptide prevents spondyloarthritis by modulating Th17/Treg imbalance in mice with curdlan-induced spondyloarthritis

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0262183
Author(s):  
Hong Ki Min ◽  
JeongWon Choi ◽  
Seon-Yeong Lee ◽  
A. Ram Lee ◽  
Byung-Moo Min ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Clinical Score ◽  
Bioactive Peptide ◽  
Vehicle Group ◽  
Αvβ3 Integrin ◽  
Increased Risk ◽  
Factor Α ◽  
Treg Differentiation

Purpose Spondyloarthritis (SpA) is a systemic inflammatory arthritis mediated mainly by interleukin (IL)-17. The vitronectin-derived bioactive peptide, VnP-16, exerts an anti-osteoporotic effect via β1 and αvβ3 integrin signaling. SpA is associated with an increased risk of osteoporosis, and we investigated the effect of VnP-16 in mice with SpA. Methods SpA was induced by curdlan in SKG ZAP-70W163C mice, which were treated with vehicle, celecoxib, VnP-16, or VnP-16+celecoxib. The clinical score, arthritis score, spondylitis score, and proinflammatory cytokine expression of the spine were evaluated by immunohistochemical staining. Type 17 helper T cell (Th17) and regulatory T cell (Treg) differentiation in the spleen was evaluated by flow cytometry and in the spine by confocal staining. Splenocyte expression of signal transducer and activator of transcription (STAT) 3 and pSTAT3 was evaluated by in vitro Western blotting. Results The clinical score was significantly reduced in the VnP16+celecoxib group. The arthritis and spondylitis scores were significantly lower in the VnP-16 and VnP16+celecoxib groups than the vehicle group. In the spine, the levels of IL-1β, IL-6, tumor necrosis factor-α, and IL-17 expression were reduced and Th17/Treg imbalance was regulated in the VnP-16 alone and VnP-16+celecoxib groups. Flow cytometry of splenocytes showed increased polarization of Tregs in the VnP-16+celecoxib group. In vitro, VnP-16 suppressed pSTAT3. Conclusions VnP-16 plus celecoxib prevented SpA progression in a mouse model by regulating the Th17/Treg imbalance and suppressing the expression of proinflammatory cytokines.

scholarly journals IL-21 Receptor Blockade Shifts the Follicular T Cell Balance and Reduces De Novo Donor-Specific Antibody Generation

2021 ◽  
Vol 12 ◽  
Author(s):  
Yeqi Nian ◽  
Zhilei Xiong ◽  
Panpan Zhan ◽  
Zhen Wang ◽  
Yang Xu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Germinal Center ◽  
De Novo ◽  
Antibody Mediated Rejection ◽  
Treg Differentiation ◽  
The Impact

Donor-specific antibodies (DSAs) play a key role in chronic kidney allograft injury. Follicular T helper (Tfh) cells trigger the humoral alloimmune response and promote DSA generation, while T-follicular regulatory (Tfr) cells inhibit antibody production by suppressing Tfh and B cells. Interleukin (IL)-21 exerts a distinct effect on Tfh and Tfr. Here, we studied whether blocking IL-21R with anti-IL-21R monoclonal antibody (αIL-21R) changes the Tfh/Tfr balance and inhibits DSA generation. First, we investigated the impact of αIL-21R on CD4+ T cell proliferation and apoptosis. The results showed that αIL-21R did not have cytotoxic effects on CD4+ T cells. Next, we examined Tfh and regulatory T cells (Tregs) in an in vitro conditioned culture model. Naïve CD4+ T cells were isolated from 3-month-old C57BL/6 mice and cultured in Tfh differentiation inducing conditions in presence of αIL-21R or isotype IgG and differentiation was evaluated by CXCR5 expression, a key Tfh marker. αIL-21R significantly inhibited Tfh differentiation. In contrast, under Treg differentiation conditions, FOXP3 expression was inhibited by IL-21. Notably, αIL-21R rescued IL-21-inhibited Treg differentiation. For in vivo investigation, a fully mismatched skin transplantation model was utilized to trigger the humoral alloimmune response. Consistently, flow cytometry revealed a reduced Tfh/Tfr ratio in recipients treated with αIL-21R. Germinal center response was evaluated by flow cytometry and lectin histochemistry. We observed that αIL-21R significantly inhibited germinal center reaction. Most importantly, DSA levels after transplantation were significantly inhibited by αIL-21R at different time points. In summary, our results demonstrate that αIL-21R shifts the Tfh/Tfr balance toward DSA inhibition. Therefore, αIL-21R may be a useful therapeutic agent to prevent chronic antibody mediated rejection after organ transplantation.

109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Nina Chu ◽  
Michael Overstreet ◽  
Ryan Gilbreth ◽  
Lori Clarke ◽  
Christina Gesse ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Dominant Negative ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

scholarly journals 228 Antagonistic pH-selective VISTA antibody SNS-101 potentiates anti-PD-1/PD-L1-induced anti-tumor immunity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A243-A243
Author(s):  
Thomas Thisted ◽  
Arnab Mukherjee ◽  
Kanam Malhotra ◽  
Zuzana Biesova ◽  
Yuliya Kleschenko ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
T Cell Activation ◽  
Drug Disposition ◽  
Checkpoint Inhibitors ◽  
Tumor Rejection ◽  
Therapeutic Window ◽  
Drug Candidate

BackgroundImmunotherapies, especially immune checkpoint inhibitors, have become a cornerstone of cancer treatment. Remarkable clinical responses have been observed blocking the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis across a spectrum of indications. However, innate and/or acquired resistance to anti-PD-1 blockade remains a major challenge. V-domain Ig suppressor of T-cell activation (VISTA) is a B7-family member, which promotes T-cell and myeloid quiescence and represents a promising target, particularly in combination with anti-PD-1/PD-L1 treatment. Recently, the interaction of VISTA with its receptor PSGL-1 was demonstrated to be significantly enhanced by the acidic tumor microenvironment (TME). As VISTA is highly expressed on myeloid cells, including those in the blood, antibodies binding VISTA at physiological pH 7.4 could result in rapid elimination from circulation through targeted-mediated drug disposition, making efficacious drug occupancy levels difficult to reach and potentially narrowing the therapeutic window. An antibody engineered to selectively bind and block VISTA at low pH in the TME may therefore be an ideal drug candidate.MethodsIn this study, fully human anti-VISTA antibodies were generated through pH-selective enrichment strategies of a yeast-based display library comprising highly diverse synthetic immune repertoires. The ‘parental’ antibodies have been extensively characterized using in vitro flow-cytometry, surface-plasmon resonance (SPR) and PSGL-1/VISTA inhibition assays in primary human CD4 and CD8 T-cells at pH 6.0 and pH 7.4. Eight parental antibodies were identified and tested for combinatorial efficacy with anti-PD-1 in vivo in human VISTA knock-in mice inoculated with syngeneic MC-38 tumors. These antibodies underwent further optimization for enhanced binding affinity at pH 6.0 and decreased binding at pH 7.4. ‘Progeny’ antibody ranking was based on the same in vitro and in vivo characterization as parental antibodies.ResultsEighty four parental antibodies were initially discovered. Flow-cytometry and SPR analysis revealed candidates displaying pH-dependent binding to endogenously expressed native VISTA on cells, and a PSGL-1/VISTA inhibition assay at pH 6.0 was run to identify and rank potent interface blockers. Eight candidate antibodies were tested in an in vivo intervention study in combination with anti-murine PD-1 demonstrating varied combinatorial efficacy with a subset leading to superior tumor rejection. Characterization of optimized progeny antibodies led to identification of anti-VISTA antibody SNS-101.ConclusionsEnrichment of highly diverse antibody libraries led to the identification of a pH-selective inhibitory anti-VISTA antibody SNS-101, which exerts excellent combinability with anti-PD-1 leading to superior anti-tumor activity in a mouse model.

scholarly journals Sunitinib Exerts In Vitro Immunomodulatory Activity on Sarcomas via Dendritic Cells and Synergizes With PD-1 Blockade

2021 ◽  
Vol 12 ◽  
Author(s):  
Darina Ocadlikova ◽  
Mariangela Lecciso ◽  
Javier Martin Broto ◽  
Katia Scotlandi ◽  
Michele Cavo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cell Lines ◽  
Effector T Cells ◽  
Sarcoma Cell ◽  
Ifn Γ ◽  
Sarcoma Cells

BackgroundHigh-grade sarcomas are a heterogeneous group of aggressive tumors arising in bone and soft tissues. After relapse, treatment options are limited. The multi-targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and inhibitor of PD-1 (anti-PD-1) nivolumab have shown antitumor activity in selected subtypes. In this study, we examine the role of TKIs and PD-1 based therapy in in vitro cocultures of sarcoma.MethodsThe human osteosarcoma (SaOS-2) and synovial sarcoma (SYO-1) cell lines were treated with sunitinib. After cell death and proliferation assessment, expression of PD-L1 was analyzed by flow cytometry. Sunitinib-treated sarcoma cells were cocultured with dendritic cells (DCs), and the phenotype of mature DCs was determined by flow cytometry. Mature DCs were cultured with autologous T cells. PD-1 expression on T cells, their proliferation, T regulatory cell (Tregs) induction and IFN-γ production, before and after nivolumab exposure, were analyzed.ResultsAlong with its anti-proliferative and direct pro-apoptotic effect on sarcoma cell lines, sunitinib prompted PD-L1 upregulation on sarcoma cells. Interestingly, sunitinib-treated sarcoma cells drive DCs to full maturation and increase their capacity to induce sarcoma-reactive T cells to produce IFN-γ. Conversely, no effect on T cell proliferation and T cell subpopulation composition was observed. Moreover, both bone and synovial sarcoma cell lines induced Tregs through DCs but sunitinib treatment completely abrogated Treg induction. Finally, sarcoma cell lines induced PD-1 upregulation on both effector T cells and Tregs when loaded into DCs, providing a rationale for using PD-1 blockade. Indeed, PD-1 blockade by nivolumab synergized with sunitinib in inducing IFN-γ-producing effector T cells.ConclusionsTaken together, our in vitro data indicate that the treatment of sarcoma cells with sunitinib can exert significant changes on immune cell subsets toward immune activation, leading to DC-based cross-priming of IFN-γ-producing effector T cells and reduced Treg induction. PD-1 blockade with nivolumab has a synergistic effect with sunitinib, supporting the use of TKI and anti-PD-1 approach in sarcomas, and perhaps in other cancers. DC-targeted drugs, including toll-like receptor 3 inhibitors and CD47 inhibitors, are under development and our preclinical model might help to better design their clinical application.

CD26+CD4+T cell counts and attack risk in interferon-treated multiple sclerosis

2005 ◽  
Vol 11 (6) ◽  
pp. 641-645 ◽  
Author(s):  
F Sellebjerg ◽  
C Ross ◽  
N Koch-Henriksen ◽  
P Soelberg Sørensen ◽  
J L Frederiksen ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Hazard Ratio ◽  
Cd4 T Cell ◽  
Immunomodulatory Treatment ◽  
Cell Counts ◽  
Increased Risk ◽  
Insufficient Response

Biomarkers that allow the identification of patients with multiple sclerosis (MS) with an insufficient response to immunomodulatory treatment would be desirable, as currently available treatments are only incompletely efficacious. Previous studies have shown that the expression of CD25, CD26 and CCR5 on T cells is altered in patients with active MS. We studied the expression of these molecules by flow cytometry in patients followed for six months during immunomodulatory treatment. In interferon (IFN)-β-treated patients, we found that the hazard ratio for developing an attack was 2.8 in patients with CD26+CD4+T cell counts above median, and this risk was independent of the risk conferred by neutralizing anti-IFN-β antibodies. CD26+CD4+T cell counts may identify patients with MS at increased risk of attack during treatment with IFN-β.

scholarly journals B7-H4 Inhibits the Development of Primary Sjögren’s Syndrome by Regulating Treg Differentiation in NOD/Ltj Mice

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Xu Zheng ◽  
Qikai Wang ◽  
Xiang Yuan ◽  
Yingbo Zhou ◽  
Hui Chu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Salivary Gland ◽  
T Cell ◽  
Salivary Glands ◽  
Inflammatory Cytokines ◽  
Mrna Levels ◽  
Lymphocyte Infiltration ◽  
Flow Cytometry Analysis ◽  
Treg Differentiation

Background. This study is aimed at exploring the role of B7-H4 in the pathogenesis of primary Sjögren’s syndrome (pSS) in NOD/Ltj mouse. Methods. B7-H4 expression in salivary glands was examined by IHC, and lymphocyte infiltration was showed by H&E. Next, anti-B7-H4 mAb or immunoglobulin isotype was injected into NOD/Ltj mice. Cytokine levels were measured by quantitative RT-PCR, and immunoglobulins were measured by ELISA. T cell subsets were analyzed by flow cytometry. Last, we treated NOD/Ltj mice with B7-H4Ig and control Ig. CD4+Foxp3+ T cells were assessed by immunohistochemistry. Two-tailed Student’s t-tests were used to detect the statistical difference in various measures between the two groups. Results. B7-H4 expression was remarkably reduced in salivary glands of NOD/Ltj mice at 15 weeks compared with the NOD/Ltj mice at 8 weeks. Anti-B7-H4 mAb treatment increased lymphocyte infiltration in salivary glands. Inflammatory cytokines including IL-12, IL-18, IL-1α, TNF-α, IFN-α, and BAFF were upregulated markedly in anti-B7-H4 mAb-treated mice compared to IgG isotype-treated mice. Flow cytometry analysis showed that anti-B7-H4 mAb-treated mice had lower levels of CD4+Foxp3+/CD4+ T cells in spleen. Moreover, Foxp3 mRNA levels of salivary glands were diminished in anti-B7-H4 mAb-treated mice. Flow cytometry analysis showed that anti-B7-H4 mAb inhibited CD4+Foxp3+/CD4+ T cell production, while B7-H4Ig would promote naïve CD4+ T into Treg differentiation. Administration with B7-H4Ig displayed significantly decreased lymphocyte infiltration in salivary glands and low levels of total IgM and IgG in serum. Analysis of inflammatory cytokines in salivary glands after B7-H4Ig treatment revealed that the mRNA levels of IL-12, IL-6, IL-18, IL-1α, TNF-α, and IFN-α were significantly downregulated in B7-H4Ig-treated mice compared to control Ig treatment. B7-H4Ig-treated mice had significantly higher levels of CD4+Foxp3+/CD4+ T cells in spleen. IHC in salivary gland revealed that CD4+Foxp3+ T cells of B7-H4Ig treatment mouse were more than control Ig treatment. Conclusions. Our findings implicate that B7-H4 has a protective role for salivary gland epithelial cells (SGECs) and therapeutic potential in the treatment of pSS.

Superior Immunogenicity of Idiotype Fab Fragments As Compared to Entire Immunoglobulin for Active Lymphoma Immunotherapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1633-1633
Author(s):  
Marcelo A. Navarrete ◽  
Benjamin Kisser ◽  
Hendrik J. Veelken
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
B Cell Lymphomas ◽  
Cd8 Cells ◽  
Fab Fragments ◽  
The Individual

Abstract Abstract 1633 Introduction: The individual collection of epitopes within the variable regions of the unique immunoglobulin expressed by every mature B-cell lymphoma (idiotype, or Id) represents a tumor-specific antigen and lends itself as a target for therapeutic vaccination strategies. Immunization with tumor Id has the capacity to elicit polyclonal antibody responses as well as CD8+ and CD4+ T cells recognizing Id-derived peptides presented on class I and class II HLA molecules, respectively. Due to a perceived low immunogenicity of lymphoma-derived Id, most Id vaccines tested in clinical trials so far have been formulated as conjugates with the strongly immunogenic carrier keyhole limpet hemocyanin (KLH). In contrast, we have consistently observed high rates of humoral and cellular anti-Id immune responses in consecutive trials of active immunization with unconjugated recombinant Fab fragments of Id in indolent B-cell lymphomas (Bertinetti et al., Cancer Res. 2006; Navarrete et al., BLOOD 2011). We therefore hypothesized that Id Fab fragment might be intrinsically more immunogenic than entire Id Ig and tested this hypothesis by comparative in vitro experiments. Methods: Monocyte-derived dendritic cells (DC) where loaded with human monoclonal IgG, papain-digested Fab fragments, Fc fragments, or recombinant lymphoma-derived Fab fragments. Functional DC phenotypes were assessed by flow cytometry of crucial maturation and activation markers. IL-10 and IL-12 was measured in DC culture supernatants by ELISA. Antigen-loaded DC where subsequently used for priming of CFSE-labeled autologous peripheral blood mononuclear cells. Stimulated T cell populations were analyzed by multicolor flow cytometry. Results: Loading of DC with Fab, Fc, IgG, or mixtures of Fab and Fc fragments did not alter surface expression of CD11c, CD80, CD83, CD86, HLA-DR, PDL-1 and PDL-2 on DC. Likewise, the various antigens did not influence the cytokine release by DC during the loading or maturation process. DC loaded with isolated Fab fragments induced significantly higher proliferation of both CD4+ and CD8+ T cells than undigested IgG. The mean proliferation rate of CD4+ cells stimulated with Fab fragments was 18.5% versus 5.6% for undigested IgG stimulation (p=0.021); proliferation rates of CD8+ cells were 14.2% versus 6.2% (p=0.034). These results were reproduced for 4 different monoclonal IgGs tested on 4 different donors. The addition of Fc fragments to Fab reduced the proliferation rates of CD4+ and CD8+ cells to 10.2% and 8.6% respectively. In addition, DC loaded with undigested IgG induced a relative increase in the number of CD25high/FoxP3+ regulatory T cells compared with Fab stimulation (8.2% versus 1.4%; p<0.01). Conclusions: Isolated Fab fragments, i.e. the Id portions that contain the individual candidate antigenic epitopes of B-cell lymphomas, prime autologous T cells in vitro more efficiently than entire IgG. This finding is consistent with the high immune response rate against recombinant unconjugated Fab fragments observed in vivo in our clinical vaccination trials. Peptide sequences shared between Ig molecules that are predominantly located in the IgG Fc fragment appear to exert an inhibitory effect on T-cell priming. In accordance with our recent in vivo data in a syngeneic mouse model of Id vaccination (Warncke et al., Cancer Immunol. Immunother. 2011), this effect may be mediated by effective activation of Treg. Fab fragments therefore appear to be the more immunogenic and therefore preferable Ig antigenic format for active anti-Id immunotherapy. Furthermore, the inhibitory effects of IgG Fc offers a potential explanation for the recently reported lack of efficacy of Id vaccination in IgG-expressing follicular lymphomas in a randomized phase III trial, in which patients with IgM-expressing lymphomas, in contrast, had a significant benefit from Id vaccination in intention-to-treat analyses (Schuster et al., JCO 2011). Disclosures: No relevant conflicts of interest to declare.

Inhibition Of Cdk2 Promotes The Generation Of Inducible CD8+ T Regulatory Cells By Modulating The Epigenetic Regulator EZH2

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 138-138
Author(s):  
Lequn Li ◽  
Nikolaos Patsoukis ◽  
Anoma Nellore ◽  
Vassiliki A. Boussiotis
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fold Increase ◽  
Treg Cells ◽  
Foxp3 Mrna ◽  
Alloreactive T Cells ◽  
Epigenetic Regulator ◽  
Treg Differentiation

Abstract Graft versus host disease (GvHD) remains the main cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation. In spite of the intense research efforts, control of GvHD remains incomplete and novel therapeutic approaches are required. Cdk2 has a central role in cell cycle re-entry of mature T lymphocytes and inhibition of Cdk2 is mandatory for induction of T cell anergy in vitro and tolerance in vivo. While Cdk2 is essential for expansion of activated T cells, it is not critical for survival of resting lymphocytes, hematopoiesis or thymocyte development. These properties make Cdk2 an attractive target for control of GvHD. To determine the effects of Cdk2 inhibition on T cell alloresponses in vivo, we used the B6D2F1 mouse model of allogeneic BMT and two different Cdk2 inhibitors, CYC202 (IC50=0.1 uM) and CYC205 (IC50=1 nM). Lethally irradiated B6D2F1(Kd) recipients were infused with bone marrow from C57BL/6(Kb) donors with (BMT) or without splenocytes (BM) and were subsequently treated with each Cdk2 inhibitor for three weeks. Treatment was administered daily during week 1, every other day on week 2, and twice a week on week 3. Effects of treatment on GvHD were assessed by body weight and survival during a 70-day period. Although BMT recipients treated with Cdk2 inhibitor displayed a transient initial weight loss, subsequently regained weight to levels comparable to control BM recipients. Furthermore, treated BMT recipient groups displayed significantly delayed GvHD mortality (p=0.0054). Recently, it was determined that inducible CD8+ Treg cells, have a central role in mediating protection from GvHD. Some immunosuppressive drugs have detrimental effects on Treg whereas others spare these cells or may even be beneficial to their proportional increase. To examine whether Cdk2 inhibitors induced Treg cells, we used GFP- T cells from Foxp3.GFP-KI mice (C57BL/6 background) as a source of T cells during BMT. Assessment of peripheral blood lymphocytes, splenocytes, peripheral lymph nodes and intestinal lymphoid cells (ILC) in BMT recipients revealed no differences in CD4+GFP+ Treg between treated and control groups. In contrast, the treated group displayed an increase of CD8+GFP+ Treg cells in these cell populations, predominantly ILC, which displayed a 5-fold increase of CD8+ Treg (p=0.05). To further investigate whether Cdk2 inhibitors had a selective effect on CD8+ Treg differentiation, we isolated CD4+GFP- and CD8+GFP- T cells from Foxp3.GFP-KI mice and subjected them to in vitro Treg polarizing with or without Cdk2 inhibitors. Inhibition of Cdk2 had almost no effect on CD4+GFP+ cells but induced a 2-4 fold increase of CD8+GFP+ cells. To determine whether Cdk2 inhibition induced its effect on CD8+ Treg differentiation by reducing the threshold of TGF-β-mediated signaling, we cultured CD8+GFP- cells with stable concentrations of Cdk2 inhibitors and decreasing concentrations of TGF-β. Cdk2 inhibition induced CD8+ Treg differentiation in the presence of TGF-β concentrations that failed to induce any significant numbers of CD8+ Treg cells when used alone. Expression of FOX family genes is regulated by transcriptional and epigenetic mechanisms. A critical epigenetic regulator of FOX transcription factors in cancer cells is the Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2), which promotes histone H3 lysine 27 trimethylation (H3K27me3) and induces epigenetic gene silencing. Cdk1 and Cdk2 phosphorylate EZH2 at Thr350 in an evolutionarily conserved motif. Phosphorylation of Thr350 is important for EZH2 recruitment and maintenance of H3K27me3 levels at EZH2-target loci. We examined whether EZH2 becomes phosphorylated in CD8+ T cells and whether Cdk2 inhibition might affect this event. Upon polarizing CD8+ T cell culture, EZH2 displayed robust phosphorylation on Thr350, which was blocked by Cdk2 inhibition. This event temporally coincided with a 44-fold increase in Foxp3 mRNA expression compared to base line levels in control T cells. These results reveal an unexpected mechanism via which Cdk2 inhibitors mediate suppression of alloreactive T cells and protection from GvHD by inducing CD8+ Treg. Because Cdk-mediated EZH2 phosphorylation is a key mechanism governing EZH2 function to regulate epigenetic silencing, Cdk2 inhibition might have additional, yet unidentified implications on gene expression programs of alloreactive T cells. Disclosures: No relevant conflicts of interest to declare.

Post-Transplant Immune Monitoring In Patients Receiving Allogeneic Stem Cell Transplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5486-5486
Author(s):  
Silvia Park ◽  
Chul Won Jung ◽  
Jun Ho Jang ◽  
Eun Suk Kang ◽  
Kihyun Kim
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Conflicts Of Interest ◽  
Immune Monitoring ◽  
Disease Relapse ◽  
Cell Secretion ◽  
Color Flow ◽  
Blood Samples ◽  
Post Transplant

Abstract Introduction There are still substantial morbidity and mortality caused by insufficient immunologic recovery after allo-HSCT. In this context, we attempt to evaluate the clinical relevance of immune monitoring in allo-HSCT recipients. Method Fifty five patients who underwent allo-HSCT between 2008 and 2012 were included. Peripheral blood samples were drawn from recipients before transplant, and on 4, 8, 12, 24, 36 and 48 weeks after transplant. Each blood samples were analyzed by multi-color flow cytometry for determining lymphocyte subsets. MNC were separated from blood specimen, and analyzed for the quantitation of Treg with the use of real-time PCR. We also examined T cell derived IFN-r by using in vitro culture, intracellular staining, and flow cytometry analysis. Results The median age was 43, and AML was the most common reason for transplantation (49.1%). Grade II or more aGVHD occurred in 36.4% of cases, and 49.1% exhibited moderate or severe cGVHD. The differences in the proportion (%) and the absolute number (/uL) of CD4+, CD8+ cells, CD4+ derived IFN-r (%), CD8+ derived IFN-r (%), and Treg (%) between the groups (Gr. II or more aGVHD (+) vs (-); moderate or severe cGVHD (+) vs (-)) were compared by Two sample t-test. Patients with Gr. II or more aGVHD showed decreased CD4+ count at 4, 8 and 12 weeks, but showed rather higher CD8+ count at 8 weeks after transplant. T-cell secretion function assessed by IFN-r (%), and Treg (%) was similar between two groups within 12 weeks after transplant. In case of cGVHD, both CD4+ and CD8+ count tended to be higher in patients with moderate or severe cGVHD, and the trends lasted for up to 48 weeks from allo-HSCT. Treg (%) was almost consistently lower throughout the period in these patients. There were 12 relapses within follow up period (median 36.1 months), and higher slope of post-transplant increase in CD8+ count and CD8 derived IFN-r were identified as protective factors for disease relapse. Conclusion In view of the results so far achieved, slow recovery of CD8 count and function might be associated with disease relapse. However, this is still a preliminary data, and warrants further evaluation. Disclosures: No relevant conflicts of interest to declare.

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