Imatinib Mesylate Impairs the Function of Human Regulatory T Cells (Tregs) and Reverses the Inhibition of Immune Responses to Imatinib-Resistant CML Cells by Tregs.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4728-4728
Author(s):  
Masahiro Ogasawara ◽  
Toshihiro Matsukawa ◽  
Yuki Katsura ◽  
Kanako Shima ◽  
Minoru Kanaya ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Cell Line ◽  
Imatinib Mesylate ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Rapid Development ◽  
Inhibitory Effect ◽  
Peripheral Tolerance ◽  
Imatinib Resistant

Abstract Abstract 4728 Imatinib mesylate (imatinib) has been widely used clinically for the treatment of CML and Ph-positive ALL patients with tremendous success. However, the effect is limited in advanced stage by rapid development of resistance to imatinib. Tregs play a pivotal role in peripheral tolerance and their impairment results in autoimmune disease and GVHD. Previous studies have revealed that imatinib inhibits the proliferation of several types of immune cells. However, immunomodulatory function of imatinib in CML remains to be elucidated. In the present study, we investigated whether imatinib exerts an immunosuppressive effect on naturally occurring Tregs in T cell responses to CML, especially imatinib-resistant CML. Peripheral blood mononuclear cells were obtained from 5 healthy volunteers with informed consent. CD4+CD25+ and CD4+CD25- T cells were isolated by magnetic cell sorting using human CD4+CD25+ T regulatory cell isolation kit (Miltenyi Biotec). Both CD4+CD25+ and CD4+CD25- T cells, pre-stimulated with anti-CD3 and anti-CD28 monoclonal antibody-coated beads for 2 days, were cultured with increasing doses of imatinib for 3 days and survival of the cells was evaluated by a WST1 tetrazolium assay. Viability decreased at higher concentrations (>10μM) of imatinib. There was no difference in the sensitivity to imatinib between CD4+CD25+ and CD4+CD25- T cells. To examine whether imatinib exerts an inhibitory effect on the proliferation of Tregs, freshly isolated CD4+CD25+ and CD4+CD25- T cells were cultured with varying concentrations of imatinib together with CD3 and CD28 stimulation for 3 days. The proliferation of these cells was inhibited by higher concentrations of imatinib in a similar fashion. Expression of FoxP3 in CD4+CD25+ Tregs was inhibited with 10μM imatinib by apporoximately 70% as shown in Figure 1. Because Treg has been shown to suppress immunity against cancer, a similar inhibitory effect is expected to occur in CML. This prompts us to examine immune responses to CML. For this purpose, we utilized a cell line (TM) established by us from a patient with CML in blastic crisis and another imatinib-resistant cell line (TM-G) generated by culturing TM with increasing concentrations of imatinib. CFSE-labeled CD4+CD25- and CD8+ responder cells were cultured for 5 days with irradiated autologous CD3- antigen presenting cells which were loaded with cell-lysates from either TM or TM-G in the presence or absence of Tregs which were pre-incubated with increasing concentrations of imatinib for 2 days. Proliferation of both CD4+CD25- and CD8+ responder cells were inhibited by Tregs which were not exposed to imatinib. On the other hand, Tregs which were exposed to imatinib had reduced inhibitory effects on the proliferation of responder cells depending on the concentration of imatinib as shown in Figure 2. These results demonstrate the immunomodulatory functions of imtinib in CML and imply that the use of this drug is a potent inhibitor of Tregs in cancer immunotherapy. Disclosures: No relevant conflicts of interest to declare.

C5a Depletion As a Novel Strategy to Inhibit T Regulatory Cells and Enhance Antigen-Specific Immune Responses

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3285-3285
Author(s):  
Suresh Veeramani ◽  
George J. Weiner
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Immune Responses ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Regulatory Cells ◽  
Complement Components ◽  
Novel Strategy

Abstract Abstract 3285 Background: The complement system has complex activity that impacts on the immune response in a broad variety of ways. The current study was designed to assess the effect of complement components, specifically C5a, on the immune regulatory cells and on the development of an antigen-specific active immune response. Methods: Myeloid dendritic cells (mDCs), enriched from healthy human peripheral blood mononuclear cells, were pulsed with antigen (tetanus toxoid) and co-cultured with autologous, enriched human CD4+ T cells in the presence of various purified complement components. The percent of CD4+ T-cells that were CD25highFoxp3+ (henceforth referred to as Tregs) was determined. The presence of cytokines in supernatant of mDCs cultured with purified complement proteins was also evaluated. In murine models, the effect of C5a on in vivo induction of Tregs and on the development of immune response to ovalbumin was determined by analyzing anti-ovalbumin antibody. This was done in C5-sufficient (B10-D2-HC1) and C5-deficient (B10-D2-HC0) mice immunized with 100 μg of ovalbumin, and in wild type C57Bl/6 mice immunized with 100 μg of ovalbumin along with either irrelevant rat IgG2a (Ova+Isotype control) or rat anti-mouse C5a antibody (Ova+anti-C5a Ab). Results: In Vitro: In Vivo: Conclusions: Presence of C5a in the immune microenvironment results in increased generation of Treg cells and leads to dampening of antigen-specific immune responses. Absence or depletion of C5a results in a drop in the Tregs and a higher antigen-specific immune response. Ongoing studies are exploring the use of C5a depletion as a novel strategy to overcome the low immunogenicity of vaccines, such as cancer vaccines. Disclosures: No relevant conflicts of interest to declare.

Resistance to the Novel Translation Inhibitor Silvestrol Is Mediated by Elevated Mcl-1 Expression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1737-1737
Author(s):  
David M. Lucas ◽  
Ellen J. Sass ◽  
Ryan B. Edwards ◽  
Li Pan ◽  
Gerard Lozanski ◽  
...  
Keyword(s):  
Drug Resistance ◽  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Multi Drug Resistance ◽  
Parental Line

Abstract Abstract 1737 Poster Board I-763 We previously reported the efficacy and B-cell selectivity of the natural product silvestrol in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), using both primary cells and B-cell lines. We also showed that silvestrol inhibits translation, resulting in rapid depletion of the short half-life protein Mcl-1 followed by mitochondrial damage and apoptosis. Cencic et al. reported that silvestrol directly blocks translation initiation by aberrantly promoting interaction of eIF4A with capped mRNA (PLoS One 2009; 4(4):e5223). However, the loss of Mcl-1 in breast and prostate cancer cell lines is delayed relative to what we observe in B-leukemias (48 hr vs. 4-6 hr in CLL and ALL cells). Additionally, silvestrol does not reduce Mcl-1 expression in normal T-cells to the same extent that it does in B-cells, potentially explaining in part the relative resistance of T-cells to this agent. We therefore investigated cell-type differences, as well as the importance of Mcl-1, in silvestrol-mediated cytotoxicity. We incubated the ALL cell line 697 with gradually increasing concentrations of silvestrol to generate a cell line (697-R) with resistance to 30 nM silvestrol (IC50 of parental 697 < 5 nM). No differences between 697-R and the parental line were detected upon detailed immunophenotyping. However, cytogenetic analysis revealed a balanced 7q;9p translocation in 697-R not present in the parental 697 cell line that may be related to the emergence of a resistant clone. We also detected no difference in expression of multi-drug resistance proteins MDR-1 and MRP, which can contribute to resistance to complex amphipathic molecules such as silvestrol. In contrast, we found that baseline Mcl-1 protein expression is strikingly increased in 697-R cells relative to the parental line, although these cells still show similar percent-wise reduction in Mcl-1 upon re-exposure to 80 nM silvestrol. To investigate whether this resistance to silvestrol is reversible, 697-R cells were maintained without silvestrol for 6 weeks (∼18 passages). During this time, viability remained near 99%. Cells were then treated with 30 nM silvestrol. Viability was 94% at 48 hr post-treatment and returned to 99% within a week, while parental 697 cells with the same treatment were completely dead. Baseline Mcl-1 levels remained elevated in 697-R even with prolonged silvestrol-free incubation. These results indicate that the resistance phenotype is not rapidly reversible, as is seen with transient upregulation of multi-drug resistance or stress-response proteins. Additionally, silvestrol moderately induces the transcription of several pro-apoptotic Bcl-2 family members and results in elevated levels of these proteins despite its translation inhibitory activity. Interestingly, no such activity is detected in silvestrol-treated normal T-cells. Together, these results support the hypothesis that in B-cells, silvestrol induces cell death by altering the balance of pro- and anti-apoptotic factors, and that increased Mcl-1 protein can force the balance back toward survival. This work further underscores the importance of Mcl-1 in silvestrol-mediated cytotoxicity. We are now investigating the mechanism of Mcl-1 upregulation in 697-R cells to identify a factor or pathway that can be targeted therapeutically to circumvent resistance. Silvestrol is currently undergoing preclinical pharmacology and toxicology investigation by the U.S. National Cancer Institute Drug Development Group at the Stage IIA level to facilitate its progression to Phase I clinical testing. Disclosures No relevant conflicts of interest to declare.

Modified Vaccinia Ankara Virus (MVA) Induces a Th1-Polarized Immune Response in Antigen-Presenting Cells (APCs).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4650-4650
Author(s):  
Christin Flechsig ◽  
Yasemin Suezer ◽  
Markus Kapp ◽  
Gerd Sutter ◽  
Hermann Einsele ◽  
...  
Keyword(s):  
Immune Response ◽  
B Cells ◽  
Immune Responses ◽  
Mononuclear Cells ◽  
Fluorescent Protein ◽  
Conflicts Of Interest ◽  
Human Peripheral Blood ◽  
Cell Transplant ◽  
Peripheral Blood Mononuclear ◽  
Leukocyte Populations

Abstract Abstract 4650 Introduction MVA is one of the most promising vaccine candidates for infectious diseases as well as for malignancies. Astonishingly, little information exists about the mechanism by which immune responses to MVA are generated. It was shown that among leukocytes - which are essential for the generation of cellular and humoral immune responses - APCs like dendritic cells, monocytes and B cells are preferentially infected. But little is known about the effects on APCs. Therefore we wanted to analyze in more detail the general effects of MVA infections on different immune cells. Methods Primary human peripheral blood mononuclear cells (PBMCs) and isolated leukocyte populations in particular monocyte derived DCs, monocytes and B cells were infected with (wildtype) wtMVA or MVA-gfp (green fluorescent protein) to verify the infection. Subsequently changes in surface markers and cytokine expression were assessed. Results Among PBMCs and specific isolated leukocyte populations, monocytes, DCs and B cells were most susceptible to MVA infection. NK cells showed a lower and T cells a very low infection rate. Surprisingly, selected monocytes were less susceptible to MVA as compared to unselected ones. This phenomenon is due to phagocytosis of other infected PBMCs by monocytes. Furthermore we could show that MVA causes a downregulation of CD14 on iDCs and monocytes as well as of CD25, CD80, and CD86 on B cells. Furthermore, there was a slight downregulation of CD1a on iDCs and mDCs and of CD80 on iDCs. On the other hand MVA caused an upregulation of HLA-DR on monocytes and additionally a slight upregulation of CD40 on iDCs. Moreover, MVA evoked a slight upregulation of CD83 on iDCs but a slight downregulation on mDCs. Above all, we could demonstrate that MVA induces an upregulation of CXCL10 in iDCs, mDCs, monocytes, and B cells, and an upregulation of TNFα, IL-6, and IL-12p70 in iDCs, mDCs, and monocytes. In addition, we revealed a downregulation of CXCL8 in monocytes as well as of IL-β in B cells. Conclusions These results suggest that MVA induces a Th1-polarized immune response in APCs. Thus, MVA seems to be an appropriate vaccine vector for antiviral immunotherapy of stem cell transplant recipients. Disclosures: No relevant conflicts of interest to declare.

CD70 Expression on HTLV-1 Infected T Cells of Carriers and ATL Patients and Its Clinical Significance

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1730-1730
Author(s):  
Izumi Masamoto ◽  
Sawako Horai ◽  
Tomohiro Kozako ◽  
Makoto Yoshimitsu ◽  
Junko Niimoto ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Surveillance Systems ◽  
Peripheral Blood Mononuclear ◽  
Tax Protein ◽  
Infected Cells

Abstract Abstract 1730 Human T-lymphotropic virus type-1(HTLV-1) is the causative agent of adult T cell leukemia/lymphoma (ATL). HTLV-1 infected T cell growth or leukemogenesis in ATL is controlled by various host immune surveillance systems. Among them, CD70 on HTLV-1 infected T cells coupled with CD27 on virus specific cytotoxic T cells has been suggested to play an important role in ATL leukemogenesis. The CD70 molecule is the only known ligand for CD27, a member of the tumor necrosis factor (TNF) receptor superfamily 7. This negative immunoregulatory pathway downregulates cytotoxic T lymphocyte activity against CD70-expressing virus infected cells. In the present study, we examined CD70 expression on primary lymphocytes of HTLV-1 carriers and ATL patients, its relationship with HTLV-1 Tax protein expression, and the effect on CTL induction. CD70 expression was higher on peripheral blood mononuclear cells (PBMCs) of HTLV-1 infected carriers compared with healthy donors (p = 0.021, n = 21, Mann-Whitney U test), and higher in ATL patients compared to carriers (p = 0.045, n = 38, Mann-Whitney U test). CD70 expression may be observed in CD4 T cells, as well as B cells, but not in CD8 T cells or monocytes. CD70 expression in CD4 T cells is related to HTLV-1 infection, because of increased detection of HTLV-1 Tax protein during over night culture of CD70-expressing cells. Experiments using an ATL cell line, in which Tax expression is inducible by doxycycline stimulation, demonstrated enhanced CD70 expression when Tax protein was induced in HTLV-1 infected cells. Anti-CD70 antibody enhanced CD107a mobilization, a marker of recent cytotoxic degranulation, in HTLV-1 Tax specific CTLs in PBMCs from asymptomatic carriers in vitro, suggesting that the CD70/CD27 pathway plays an important role in the immune response to HTLV-1 infection in carriers, as well as ATL patients. Disclosures: No relevant conflicts of interest to declare.

Robust Induction Of Th1-Like γδ T Cells With Anti-Myeloma Activity By Lenalidomide In Combination With HMB-PP As Well As Zoledronic Acid

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 129-129
Author(s):  
Takeshi Harada ◽  
Qu Cui ◽  
Shingen Nakamura ◽  
Hirokazu Miki ◽  
Asuka Oda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Zoledronic Acid ◽  
Cytotoxic Activity ◽  
Immune Cells ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Γδ T Cells ◽  
Bone Marrow Microenvironment

Abstract Multiple myeloma (MM) still remains incurable even with the implementation of novel therapeutic modalities, leading to the idea to develop various forms of immunotherapies. In this regard, γδ T cells bearing Vγ9Vδ2 TCR expanded from peripheral blood mononuclear cells (PBMCs) have attracted attention as potent effectors available in a novel immunotherapy against MM. Human Vγ9Vδ2 γδ T cells can be expanded ex vivo by aminobisphosphonates in combination with IL-2, and effectively target and impair MM cells. However, MM cells appear to protect themselves from external insults by immune cells in a unique bone marrow microenvironment created by the accumulation of mesenchymal stem cells/bone marrow stromal cells (BMSCs) with defective osteoblastic differentiation and acid-producing osteoclasts. To improve the therapeutic efficacy of γδ T cells, therefore, we need to develop a maneuver to effectively enhance the expansion and activity of γδ T cells while disrupting the MM cell-bone marrow interaction. Lenalidomide (Len), a novel immunomodulatory anti-MM agent, shows pivotal anti-MM activity by targeting immune cells as well as the interaction of MM cells and their surrounding cells in the bone marrow. The present study was undertaken to explore the efficacy of Len in combination with zoledronic acid (Zol) or a precursor of isopentenyl pyrophosphate (IPP) (E)-4 hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), a microbial antigen for Vγ9Vδ2 TCR, on the induction and expansion of Th1-like γδ T cells with enhanced cytotoxic activity against MM cells in the skewed bone marrow microenvironment in MM. When combined with Zol (1μM), clinically relevant doses of Len (around 1 μM) substantially expanded γδ T cells from PBMCs to the levels similar to IL-2 (100 U/ml). Len was able to expand γδ T cells more robustly in combination with HMB-PP (1 μM) than Zol from PBMCs from the majority of normal donors. However, Len alone did not show any significant effects on γδ T cell expansion and activation, suggesting a costimulatory role of Len on Zol or HMB-PP-primed γδ T cells. The surface expression of LFA-1, and the cytotoxicity-associated molecules NKG2D, DNAX accessory molecule-1 (DNAM-1; CD226) and TRAIL were up-regulated in the expanded γδ T cells. Although functional diversity has been demonstrated in γδ T cells expanded by various stimuli, Len in combination with either Zol or HMB-PP enhanced intracellular IFN-γ along with the surface NKG2D but not Foxp3 in γδ T cells at higher levels than IL-2, suggesting robust induction of Th1-like γδ T cells by Len. Importantly, γδ T cells expanded with the combinatory treatments with Len and Zol or HMB-PP exerted potent cytotoxic activity against MM cells but not normal cells surrounding MM cells in bone marrow samples from patients with MM. Such treatments with Len was able to maintain the cytotoxic activity of the γδ T cells against MM cells in acidic conditions with lactic acid, and restored their anti-MM activity blunted in the presence of BMSCs. Interestingly, the expanded γδ T cells markedly suppressed the colony formation in semi-solid methylcellulose assays of RPMI8226 and KMS-11 cells [81±1 (mean ± SD) vs. 0±0 and 40±1 vs. 16±4 colonies/dish, respectively, p<0.01], and decreased in size their side populations, suggesting targeting a drug-resistant clonogenic MM cells. These results collectively demonstrate that Len and HMB-PP as well as Zol are an effective combination for ex vivo expansion of Th1-like γδ T cells with potent anti-MM activity, and suggest that Len in combination with Zol may maintain their in vivo anti-MM activity in the bone marrow where MM cells reside. The present results warrant further study on Len-based immunotherapy with γδ T cells. Disclosures: No relevant conflicts of interest to declare.

Interleukin-2 (hrIL2) Primed T cells display Reduced Alloproliferative Capacity In Vitro Due To Induction Of FOXP3+ Regulatory Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5433-5433
Author(s):  
Dimitra Kokkinou ◽  
Panagiota Stamou ◽  
Angeliki Vittoraki ◽  
Anne-Lise De Lastic ◽  
Spyros Chondropoulos ◽  
...  
Keyword(s):  
T Cells ◽  
Low Dose ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Interleukin 2 ◽  
Regulatory Cells ◽  
Transplant Tolerance ◽  
Immune Exhaustion ◽  
The Impact

Abstract Introduction Prophylactic donor lymphocyte infusions (pDLI) after allogeneic transplantation contribute to immune restoration and reduce viral infections. Furthermore, we have recently shown that pDLI in patients with high risk leukemia significantly reduces the relapse rate, however, they were associated with a relatively high incidence of Graft versus Host Disease (GvHD)(BBMT 2013;19:75-81). Strategies to minimize GvHD without compromising the effect of pDLI against leukemia are needed. IL2 plays dual role in immune responses, contributing to both the generation of effector T cells and the maintenance of regulatory T cells (Tregs). Recently, low dose interleukin-2 (IL-2) therapy has been advanced as a potential immune modulator able to modulate the immune response to aid transplant tolerance and to suppress GvHD through expansion of Tregs (N Engl J Med. 2011; 2055-66). We investigated the impact of priming DLI with low dose IL2 on the proliferative responses to allo-stimulation in vitro. Methods CD3+ T cells purified from healthy individuals by MACS negative selection were primed (p-T cells) or unprimed (np-T cells), with or without (control) 100 U/ml hrIL2 (Proleukin, Novartis) for 7 days. Composition of T-cell cultures was analyzed by flow cytometry for: a) the percentage of T regulatory cells (CD4+/CD25high/Foxp3+/Helios+, b) their differentiation (CD28/CD27), c) their immune exhaustion (Programmed cell death 1, PD1). In vitro alloproliferative capacity of the p-T cells was analyzed with CFSE cell proliferation assay by using them as responder cells in mixed lymphocyte cultures (MLC), with irradiated allo-PB mononuclear cells as stimulators. Results In vitro priming of T-cells with IL-2 (p-T cells) in contrast to np-T or control cells: 1) increase the numbers of CD4+CD25highFoxp3+/Helios+ cells (n=8, 3.3%±0.7 mean±SEM vs 1.01%±0.22, p=0.004 και 1.4%±0.42, p=0.006). Increased levels of Foxp3 expression was also confirmed by Real Time PCR (n=2,1.25AU±0.15 vs 0.29AU±0.04, p=0.028 και 0.26AU±0.07, p=0.024). 2) did not affect the proportion of CD28+/CD27+ non late-differentiated cells (n=3, 60%±0.15 vs61%±0.04, p=0.91 και 59%±0.08, p=0.024). 3) did not cause immune exhaustion through PD1 expression (n=6, 13.3%±1.9 vs 8.1%±2.1, p=0.76, και 14%±2.2, p=0.68). 4) significantly decreases their response rate to allo-stimulus in MLC (n=8, 45%±0.5 vs65%±0.2, p=0.006 και 64%±0.2, p=0.008). The p-T cells regained their alloproliferative capacity after FACS-sorting removal of CD4+/CD25high Tregs. Conclusions Our results show that ex vivo priming of T cells with low dose of IL-2 reduces their in vitro alloproliferative capacity. This reduction is not due to late differentiation or immune – exhaustion of T cells but to selective induction of Foxp3+ cells with immunomodulatory properties in the culture. It remains to be seen whether IL2-primed DLI is safe and effective in transplant patients. Disclosures: No relevant conflicts of interest to declare.

Sipuleucel-T (sip-T) induced lytic CD8+ T cell responses to target antigens in men with hormone-sensitive and castration-resistant prostate cancer (CRPC).

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 162-162
Author(s):  
Emmanuel S. Antonarakis ◽  
David I. Quinn ◽  
Adam S. Kibel ◽  
Daniel Peter Petrylak ◽  
Tuyen Vu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
T Cell Responses ◽  
Cell Responses ◽  
Hormone Sensitive ◽  
Ctl Activity

162 Background: Sip-T is an FDA-approved immunotherapy for patients (pts) with asymptomatic or minimally symptomatic metastatic CRPC. Sip-T is manufactured from autologous peripheral blood mononuclear cells cultured with the immunogen PA2024, a fusion antigen of prostatic acid phosphatase (PAP) conjugated to granulocyte macrophage colony-stimulating factor. After sip-T, antibody and T cell responses to PA2024 and/or PAP correlate with improved survival. To further elucidate the mechanism of sip-T–induced immune responses, we evaluated the proliferative and lytic ability of PA2024- and PAP-specific CD8+ T cells. Methods: Mononuclear blood cells were labeled with the membrane dye carboxyfluorescein succinimidyl ester (CFSE) and cultured with PA2024 or PAP. In vitro proliferative and lytic CD8+ (cytotoxic T lymphocyte [CTL]) T cell responses to these antigens were evaluated by flow cytometry. For proliferation, progressive dilution of CFSE was measured. For CTL activity, the loss of intracellular granzyme B (GzB), indicating exocytosis of this apoptosis-mediating enzyme, was assessed. Samples were from 2 sip-T clinical trials STAND (NCT01431391) and STRIDE (NCT01981122), hormone-sensitive and CRPC pts, respectively. Results: Six wk after sip-T administration, CD8+ PAP- and PA2024-specific responses were observed (n=14 pts assessed). The magnitude of PA2024-specific CD8+ proliferative responses was greater than that for PAP-specific responses. CD8+ T cells from a subset of pts who exhibited PA2024- and/or PAP-specific proliferative responses were assessed for lytic ability. After in vitro antigen stimulation, CTL activity in all evaluated samples (n=14, PA2024; n=13, PAP) was demonstrated by a significant decrease (p<0.05) in intracellular GzB relative to a no-antigen control. Conclusions: Sip-T induced CD8+ CTL proliferation against the target antigens PAP and PA2024. Moreover, antigen-specific CTL activity provides the first direct evidence that sip-T can induce tumor cell lysis. These antigen-specific CD8+ lytic abilities were observed within 6 wk following sip-T, suggesting rapidly generated immune responses. Clinical trial information: NCT01431391; NCT01981122.

scholarly journals CD4+ CD25+ Regulatory T Cells Control T Helper Cell Type 1 Responses to Foreign Antigens Induced by Mature Dendritic Cells In Vivo

2003 ◽  
Vol 198 (2) ◽  
pp. 259-266 ◽  
Author(s):  
Guillaume Oldenhove ◽  
Magali de Heusch ◽  
Georgette Urbain-Vansanten ◽  
Jacques Urbain ◽  
Charlie Maliszewski ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
Peripheral Tolerance ◽  
T Helper Cell ◽  
Helper Cell ◽  
T Helper

Recent evidence suggests that in addition to their well known stimulatory properties, dendritic cells (DCs) may play a major role in peripheral tolerance. It is still unclear whether a distinct subtype or activation status of DC exists that promotes the differentiation of suppressor rather than effector T cells from naive precursors. In this work, we tested whether the naturally occurring CD4+ CD25+ regulatory T cells (Treg) may control immune responses induced by DCs in vivo. We characterized the immune response induced by adoptive transfer of antigen-pulsed mature DCs into mice depleted or not of CD25+ cells. We found that the development of major histocompatibility complex class I and II–restricted interferon γ–producing cells was consistently enhanced in the absence of Treg. By contrast, T helper cell (Th)2 priming was down-regulated in the same conditions. This regulation was independent of interleukin 10 production by DCs. Of note, splenic DCs incubated in vitro with Toll-like receptor ligands (lipopolysaccharide or CpG) activated immune responses that remained sensitive to Treg function. Our data further show that mature DCs induced higher cytotoxic activity in CD25-depleted recipients as compared with untreated hosts. We conclude that Treg naturally exert a negative feedback mechanism on Th1-type responses induced by mature DCs in vivo.

scholarly journals Multifunctional T Cell Response to DosR and Rpf Antigens Is Associated with Protection in Long-Term Mycobacterium tuberculosis-Infected Individuals in Colombia

2016 ◽  
Vol 23 (10) ◽  
pp. 813-824 ◽  
Author(s):  
Leonar Arroyo ◽  
Mauricio Rojas ◽  
Kees L. M. C. Franken ◽  
Tom H. M. Ottenhoff ◽  
Luis F. Barrera
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
T Cell ◽  
Immune Responses ◽  
Mononuclear Cells ◽  
T Cell Response ◽  
Short Term ◽  
Content Type ◽  
Long Term Culture

ABSTRACTMultifunctional T cells have been shown to be protective in chronic viral infections. In mycobacterial infections, however, evidence for a protective role of multifunctional T cells remains inconclusive. Short-term cultures of peripheral blood mononuclear cells stimulated with theMycobacterium tuberculosisRD1 antigens 6-kDa early secretory antigenic target (ESAT6) and 10-kDa culture filtrate antigen (CFP10), which are induced in the early infection phase, have been mainly used to assess T cell multifunctionality, although long-term culture assays have been proposed to be more sensitive than short-term assays for assessment of memory T cells, which are essential for long-term immunity. Here we used a long-term culture assay system to study the T cell immune responses to theM. tuberculosislatency-associated DosR antigens and reactivation-associated Rpf antigens, compared to ESAT6 and CFP10, in patients with pulmonary tuberculosis (PTB) and household contacts of PTB patients with long-term latent tuberculosis infection (ltLTBI), in a community in whichM. tuberculosisis endemic. Our results showed that the DosR antigens Rv1737c (narK2) and Rv2029c (pfkB) and the Rv2389c (rpfD) antigen ofM. tuberculosisinduced higher frequencies of CD4+or CD8+mono- or bifunctional (but not multifunctional) T cells producing interferon gamma (IFN-γ) and/or tumor necrosis alpha (TNF-α) in ltLTBI, compared to PTB. Moreover, the frequencies of CD4+and/or CD8+T cells with a CD45RO+CD27+phenotype were higher in ltLTBI than in PTB. Thus, the immune responses to selected DosR and Rpf antigens may be associated with long-term latency, correlating with protection fromM. tuberculosisreactivation in ltLTBI. Further study of the functional and memory phenotypes may contribute to further discrimination between the different states ofM. tuberculosisinfections.

scholarly journals Development of Potency Assays for Quality Assessment of an Advanced Therapy Medicinal Product: Mitomycin C-Induced Peripheral Blood Mononuclear Cell (MIC) Product

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5689-5689
Author(s):  
Ming NI ◽  
Lei Wang ◽  
Christian Kleist ◽  
Peter Terness ◽  
Gerhard Opelz ◽  
...  
Keyword(s):  
T Cells ◽  
Quality Assessment ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Membrane Surface ◽  
Advanced Therapy Medicinal Product ◽  
Peripheral Blood Mononuclear ◽  
Advanced Therapy

Abstract Introduction: Quality assessment in terms of product specificity and product potency is obligatory and strictly required by EMA and FDA to provide a platform for analysis of product comparability, stability and compatibility, and for clinical development to help predict product clinical efficacy. Mitomycin-treated donor peripheral blood mononuclear cells (MICs) could induce donor-specific tolerance via generation of recipient-derived tolerogenic dendritic cells (tDCs). To evaluate the immunomodulating capacity of the MIC product, we assessed the phenotype of tDCs, the immunosuppressive capacity of tDCs on allo-reactive CD4+ and CD8+ T cells as well as on CMV-specific CD8+ T cells. Methods: For standardization of the potency assay, third-party PBMCs positive for HLA-A2 and anti-CMV IgG were used. Immature DCs (iDCs) were generated 3 days before MIC production. MICs were generated under Good Manufacturing Practice (GMP) conditions. Each batch of MICs was introduced to iDCs at different ratios (1:0, 1:1, 1:10, 1:20) for a two-hour interaction followed by adding a DC maturation cocktail for overnight co-culture. Thereafter tDCs were purified by magnetic negative separation. The morphology of tDCs was observed by microscopy. The expression of HLA-DR, CD80, CD83, CD86 and CD103 was analyzed by flow cytometry. The inhibitory capacity on allo-reactive T cells and virus specific T cells was determined by mixed lymphocyte reaction (MLR) assay, by ELISpot assay and by Tetramer staining assay after one week of expansion in mixed lymphocyte peptide culture (MLPC). Results: In light microscopy (magnification: x 40), tDCs showed a relative smooth membrane surface. While the conventional mDCs were significantly larger in size with rough surface, richer ruffles on the cell membrane, and bigger, longer protrusions or pseudopodia. MIC products could inhibit the expression of costimulatory molecules on tDCs with an inhibition of 41% of CD80, 27% of CD83 and 23% of CD86. In parallel, the inhibitory marker CD103 was up-regulated about 65% on tDCs. Functionally, both tDCs and MICs could inhibit the IFN-γ secretion by CMV-specific CD8+ T cells, respectively. Moreover, the proliferation of allo-reactive T cells and CMV-specific T cells could be inhibited by tDCs and MICs. Conclusions: In summary, the potency assays, including the measurement of physicochemical parameters, the morphology and marker expression of tDCs, as well as the biologic characterization, the functionally immunosuppressive capacity of tDCs, comprise valuable parameters for the evaluation of clinically used advanced therapeutic cellular products. Disclosures No relevant conflicts of interest to declare.

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