Differences between Exhausted CD8+ T cells in Hepatocellular Carcinoma Patients with and without Uremia

2020 ◽  
Author(s):  
Xiaohong Chen ◽  
Jianzhou Zou ◽  
Bo Shen ◽  
Wenlv Lv ◽  
Xuesen Cao ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
Cd8 T Cells ◽  
Ifn Γ ◽  
Uremic Patients ◽  
Anti Tumor Activity

Purpose: To explore the differences between exhausted CD8+ T cells in HCC patients with and without uremia. Methods: We enrolled 45 uremic patients who were recently diagnosed with HCC into the HCC & uremia cohort. We also enrolled similar patients with HCC but without uremia; this was the HCC only cohort. Lymphocytes were obtained from the two cohorts and exhausted CD8+ T cells, comprising PD-1+CD8+, TIM-3+CD8+, and LAG-3+CD8+ T cells, were sorted and expanded in vitro. Results: The proportions of PD-1+CD8+, TIM-3+CD8+, and LAG-3+CD8+ T cells after expansion were significantly higher in the HCC only cohort as compared to those in the HCC & uremia cohort. CD8+ T cells expressing PD-1, TIM-3, or LAG-3 showed increased tumor reactivity and release of IFN-γ in vitro; however, these cells demonstrated weaker anti-tumor activity in HCC & uremia patients than those in HCC patients without uremia. Among the expanded lymphocytes, only the decreased proportion of PD-1+CD8+ T cells correlated with the HCC & uremia cohort (OR: 2.731, p=0.009). Conclusions: Peripheral CD8+ T cells expressing PD-1, TIM-3, or LAG-3 from the HCC & uremia cohort were dysfunctional in vitro. Among these populations, PD-1+CD8+ T cells were the most predominant in HCC patients with uremia.

scholarly journals The Immunomodulatory Effect of Triptolide on Mesenchymal Stem Cells in Vitro

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5011-5011
Author(s):  
Haiping He ◽  
Atsuko Takahashi ◽  
Yuki Yamamoto ◽  
Akiko Hori ◽  
Yuta Miharu ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Complete Medium ◽  
Surface Markers ◽  
Rt Pcr ◽  
Tnf Α ◽  
Ifn Γ

Background: Mesenchymal stromal cells (MSC) are known to have the immunosuppressive ability and have been applied in clinic to treat acute graft-versus-host disease (GVHD), as one of severe complications after hematopoietic stem cells transplantation (HSCT) in Japan. However, MSC are activated to suppress the immune system only upon the stimulation of inflammatory cytokines and the clinical results of MSC therapies for acute GVHD are varied. It is ideal that MSC are primed to be activated and ready to suppress the immunity (=priming) before administration in vivo. Triptolide (TPL) is a diterpene triepoxide purified from a Chinese herb - Tripterygium Wilfordii Hook F (TWHF). It has been shown to possess anti-inflammatory and immunosuppressive properties in vitro. In this study, we aim to use TPL as the activator for umbilical cord-derived MSC (UC-MSC) to entry stronger immunosuppressive status. Methods: The proliferation of UC-MSC with TPL at the indicated concentrations for different time of 24, 48, 72, and 96 hours. Cell counting kit-8(CCK-8) was added in the culture medium to detect cell toxicity and the absorbance was measured using microplate reader. Flow cytometry was used to identify the MSC surface markers expression. TPL-primed UC-MSC were once replaced with fresh medium and co-culture with mixed lymphocyte reaction (MLR) consisted with mononuclear cells (MNCs) stained with CFSE and irradiated allogenic dendritic cell line (PMDC05) in RPMI 1640 medium supplemented with 10 % FBS (complete medium). IDO-1, SOD1, and TGF-β gene expression in TPL-primed UC-MSC and UC-MSC induced by 10 ng/ml IFN-γ and/or 15 ng/ml TNF-α were evaluated by RT-PCR. PDL1 and PDL2 expression in TPL-primed UC-MSC and UC-MSC in response to IFN-γ and/or TNF-α were checked by Flowjo. Results: Exposure of TPL for UC-MSC for 72hour at the concentration above 0.1 μM resulted in the cell damage significantly. Therefore, we added TPL in UC-MSC at 0.01μM of TPL for up to 48 hours, then washed thourouphly for the following culture for experiments. To evaluate the influence of TPL on the surface markers of UC-MSC, we cultured UC-MSC for 4 hours in complete medium following culture with 0.01μM of TPL for 20 hours (TPL-primed UC-MSC). TPL-primed UC-MSC revealed positive for CD105, CD73, and CD90, negative for CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules as same as the non-primed UC-MSC. In MLR suppression by UC-MSC, the TPL-primed UC-MSC activity revealed stronger anti-proliferative effect on the CD4+ and CD8+ T cells activated by allogeneic DC than those of non-primed UC-MSC in MLR. Furthermore, the TPL-primed UC-MSC promoted the expression of IDO-1, SOD1 and TGF-β in response to IFN-γ+/-TNF-α by RT-PCR and enhanced the expression of PD-L1 by FACS analysis. Discussion:In this study, we found the TPL-primed UC-MSC showed stronger antiproliferative potency on CD4+ and CD8+ T cells compared with non-primed UC-MSC. TPL-primed UC-MSC promoted the expression of IDO-1, SOD1 and TGF-β stimulated by IFN-γ+/-TNF-α, although TPL alone did not induce these factors. Furthermore, we found that the PD1 ligand (PD-L1) was induced in TPL-primed UC-MSC, likely IFN-γ enhanced the PD-L1 expression, evaluated by flowcytometry. These results suggested that TPL-primed UC-MSC seemed more sensitive to be activated as the immunosuppressant. Here, we firstly report the new function of TPL to induce the upregulation of immunosuppressive effect, although the mechanisms of TPL inhibition to MSC need to be explore. Conclusively, TPL-primed UC-MSC might be applied for the immunosuppressive inducer of MSC. Figure Disclosures He: SASAGAWA Medical Scholarship: Research Funding; IMSUT Joint Research Project: Research Funding. Nagamura:AMED: Research Funding. Tojo:AMED: Research Funding; Torii Pharmaceutical: Research Funding. Nagamura-Inoue:AMED: Research Funding.

scholarly journals The effect of Kefir on The Immune Response of Healthy Volunteers In Vitro

2017 ◽  
Vol 3 (2) ◽  
pp. 28
Author(s):  
Desie Dwi Wisudanti
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Immune Responses ◽  
Healthy Volunteers ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Fermented Milk ◽  
Bioactive Components ◽  
Ifn Γ

Kefir is a functional foodstuff of probiotics, made from fermented milk with kefir grains containing various types of beneficial bacteria and yeast. There have been many studies on the effects of oral kefir on the immune system, but few studies have shown the effect of bioactive components from kefir (peptides and exopolysaccharides/ kefiran), on immune responses. The purpose of this study was to prove the effect of kefir supernatant from milk goat on healthy immune volunteer response in vitro. The study was conducted on 15 healthy volunteers, then isolated PBMC from whole blood, then divided into 5 groups (K-, P1, P2, P3 and P4) before culture was done for 4 days. The harvested cells from culture were examined for the percentage of CD4+ T cells, CD8+ T cells, IFN-γ, IL-4 using flowsitometry and IL-2 levels, IL-10 using the ELISA method. The results obtained that kefir do not affect the percentage of CD4+ T cells and CD8+ T cells. The higher the concentration of kefir given, the higher levels of secreted IFN- γ and IL-4, but a decrease in IL-2 levels. Significant enhancement occurred at levels of IL-10 culture PBMC given kefir with various concentrations (p <0.01), especially at concentrations of 1%. These results also show the important effects of kefir bioactive components on immune responses. The conclusion of this study is that kefir can improve the immune response, through stimulation of IL-10 secretion in vitro.

Adoptive T Cell Therapy of Human CMV Infection in Immunocompromised Hosts: In Vitro Generation of CMV-Specific T Cells from Naïve Precursors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 206-206 ◽  
Author(s):  
Sonja Schmucker ◽  
Mario Assenmacher ◽  
Jurgen Schmitz ◽  
Anne Richter
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Peptide Pool ◽  
Adoptive T Cell Therapy ◽  
T Cell Therapy ◽  
A Cell ◽  
Ifn Γ

Abstract Adoptive transfer of virus-specific T cells is a promising therapy for the treatment of infections in immunocompromised patients. Virus-specific T cells can readily be obtained from antigen-experienced, but not naïve donors. In this study we describe a cell culture system for the in vitro generation of CMV-specific T cells from naive T cells derived from CMV-seronegative donors. We isolated naïve T cells by magnetic depletion of non-T cells, CD25+ regulatory T cells, and CD45RO+ effector and memory T cells from peripheral blood mononuclear cells (PBMC) of CMV-seronegative donors. These naïve T cells were co-cultured with autologous mature monocyte-derived DC (MoDC) loaded with a pool of overlapping peptides from the CMV protein pp65. CD3-depleted autologous PBMC were used as feeder cells and CD28 antibody, IL-2, IL-7, and IL-15 were added to the culture. Already only 9–13 days after starting the priming culture, frequencies of 0.0024% and 0.009% pp65495–503/A2-tetramer+ cells among CD8+ T cells were found for 2 HLA-A2+ blood donors. In contrast pp65495–503/A2-tetramer+ T cells were not detectable when naive T cells were cultured with unpulsed MoDC. Tetramers are suitable tools for the identification of antigen-specific T cells but are restricted to single epitopes of mainly CD8+ T cells. To analyze primed CD4+ T cells as well as CD8+ T cells having specificities other than for the peptide pp65495–503, we looked for upregulation of the activation marker CD137 after a second stimulation and found increased frequencies of CD137+ CD4+ T cells as well as CD137+ CD8+ T cells in the pp65-primed cell cultures only when restimulated with the peptide pool of pp65. Because IFN-γ is important for the control of CMV infection, we studied the capability of the in vitro primed pp65-specific CD4+ and CD8+ T cells to produce this cytokine. Restimulation of the T cells with pp65 peptide pool induced IFN-γ secretion in up to 3.9% of the CD8+ T cells and up to 3.8% of the CD4+ T cells in each of six donors tested. No specific IFN-γ production was detected after restimulation with an irrelevant IE-1 peptide pool. As expected the frequency of pp65-specific T cells in the priming cultures is low. For generation of T cell lines, we magnetically enrich pp65- specific T cells according to their IFN-γ secretion using the cytokine secretion assay technology. After further cultivation for 2 weeks the antigen-specificity of the expanded T cells was again evaluated. Only if restimulated with the pp65 peptide pool 56.6% of the CD4+ T cells showed upregulated expression of the activation marker CD154 (CD40L). Cytokine analysis of the cells revealed IFN-γ production in 40.2% of the CD4+ T cells, of which 36% co-expressed IL-2, indicating the functionality of the in vitro primed and expanded T cells. In conclusion, we established a cell culture system for in vitro priming of CMV-specific CD4+ and CD8+ T cells derived from peripheral blood of donors not infected by CMV. This should extend the application of adoptive T cell therapy to patients for whom immune donors are not available.

Phagocytosis May Be a Regulatory Mechanism for Myeloid Dendritic Cells to Terminate Type 1 CD8+ T Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1654-1654
Author(s):  
Young-June Kim ◽  
Hal E. Broxmeyer
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Cd8 T Cells ◽  
Phagocytic Activity ◽  
Regulatory Mechanism ◽  
Gm Csf ◽  
T Cell Immune Responses ◽  
Ifn Γ

Abstract Abstract 1654 Poster Board I-680 CD8+ cytotoxic T cells are often ‘exhausted’ by programmed death-1 (PD-1) signaling, and subsequently the functions of these cells are terminated especially in a tumor environment or upon chronic HIV or HCV infection. Subsets of myeloid cells referred to as myeloid derived suppressor cells (MDSC) or regulatory dendritic cells (DCs) have been implicated in inducing exhaustion or termination of effector CD8+ T cells. To this end, we developed various myeloid-derived dendritic cell (DC) types in vitro from human CD14+ monocytes using M-CSF or GM-CSF in the presence of IL-4 with/without other cytokines, and characterized these DCs with respect to their capacity to induce PD-1 expression on and exhaustion of CD8+ T cells. We then assessed their impact on longevity of CD8+ T cells following coculture. Myeloid DCs developed in vitro with M-CSF and IL-4 for 5 days (referred to as M-DC) did not express ligand for PD-1 (PD-L1) nor did they induce PD-1 on CD8+ T cells. Thus, using M-DCs as starting cells, we sought determinant factors that could modulate M-DCs to express PD-L1 and thereby induce exhaustion of CD8+ T cells. In order to better monitor exhaustion processes, we incubated human peripheral CD8+ T cells for 5 days in the presence of IL-15, an important cytokine for maintaining viability, before coculture. M-DCs showed little impact on exhaustion or longevity of the CD8+ T cells. IL-10 converted M-DC into a distinct myeloid DC subset (referred to as M-DC/IL-10) with an ability to express PD-L1 as well as to induce PD-1 on cocultured CD8+ T cells. M-DC/IL-10 cells markedly suppressed proliferation of cocultured CD8+ T cells. M-DC/IL-10 cells were morphologically unique with many granules and filamentous structures around the cell periphery. These IL-10 effects on M-DC were completely abrogated in the presence of TNF-á. M-DC/IL-10 cells could be further differentiated into another myeloid DC subset in the presence of IFN-γ (referred to as M-DC/IL-10/IFN-γ) with an ability to express even higher levels of PD-L1 compared to M-DC/IL-10 cells. The most remarkable effect of M-DC/IL-10/IFN-γ cells on cocultured CD8+ T cells was a dramatic loss of CD8+ T cells. Light and confocal microscopic observations indicated that loss of CD8+ T cells was due to phagocytosis by M-DC/IL-10/IFN-γ cells. As IFN-γ, a type 1 cytokine which is induced in CD8+ T cells by IL-12 is essential for phagocytosis, we tested whether IL-12 treatment of CD8+ T cells could further enhance phagocytosis induced by M-DC/IL-10/IFN-γ cells. Indeed, IL-12 treatment greatly increased numbers of phagocytosed CD8+ T cells. In contrast, IL-4 treated CD8+ T cells became resistant to phagocytosis, suggesting IFN-γ producing (type1) CD8+ T cells may be primary target cells for the M-DC/IL-10 cells mediated phagocytosis. CD4+ T cells were not as susceptible as CD8+ T cells to phagocytosis. We failed to detect such phagocytic activity induced by prototype DCs generated with GM-CSF and IL-4. Phagocytic activity was not inhibited by various arginase-1 inhibitors suggesting that nitric oxide signaling may not mediate phagocytic activity. Neutralizing antibody to PD-L1 slightly but significantly lowered phagocytic activity suggesting that PD-L1/PD-1 interaction may be partially involved in this process. Myeloid DCs are thought to be immunogenic, actively inducing T cell immune responses. Our results demonstrate that myeloid DCs may play suppressive roles as well through induction of phagocytic activity, especially against IFN-γ producing CD8+ T cells. This may serve as a regulatory mechanism for type 1 CD8+ T cell immune responses in an IL-10 enriched microenvironment. Disclosures No relevant conflicts of interest to declare.

Donor Immunization with WT1 Peptide Augments Anti-Leukemic Activity After MHC-Matched Bone Marrow Transplantation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1896-1896
Author(s):  
Holbrook E Kohrt ◽  
Antonia MS Mueller ◽  
Jeanette B Baker ◽  
Matthew J Goldstein ◽  
Evan Newell ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Tumor Antigen ◽  
Cd4 T Cell ◽  
Leukemia Cells ◽  
Peptide Vaccination ◽  
Tumor Bearing ◽  
Ifn Γ ◽  
Anti Tumor Activity

Abstract Abstract 1896 The curative potential of MHC-matched allogeneic bone marrow transplantation (BMT) is in part due to immunologic graft-versus-tumor (GvT) reactions mediated by donor T cells that recognize host minor histocompatibility antigens. Immunization with leukemia-associated antigens, such as Wilm's Tumor 1 (WT1) peptides, induces a T cell population that is tumor antigen specific. We determined whether BMT combined with immunotherapy using WT1 peptide vaccination of donors induced more potent anti-tumor activity when combined with allotransplantation. WT1 peptide vaccinations of healthy syngeneic or allogeneic donor mice with a 9-mer WT1 peptide (amino acids 126–134, the WT1 9-mer which has the highest binding affinity for H-2Db) and Incomplete Freund's Adjuvant induced CD8+ T cells that were specifically reactive to WT1-expressing FBL3 leukemia cells. We found that compared to vaccination with IFA alone, four weekly WT1 vaccinations induced an increased percentage of WT1-tetramer+CD8 T-cells (0.15% vs. 1%) in the peripheral blood 28 days following the first vaccination (Figure A *p<.001). CD8 T-cells producing IFN-γ+ after co-culture with tumor cells were similarly increased (0.11% vs. 13.6%) at this timepoint (Figure B *p<.001). They were CD44hi suggesting a memory phenotype, specifically reactive to WT1-expressing tumor (FBL3 and not H11), and increased in a vaccination dose-dependent fashion (Figure A and B). Four weekly WT1 vaccinations prevented tumor growth in donors following intravenous leukemia challenge. In contrast, in tumor-bearing mice, WT1 vaccinations failed to induce WT1-tetramer+ or IFN-γ+ CD8 T-cells and were ineffective as a therapeutic vaccine based on intensity of bioluminescence from luciferase-labeled FBL3 leukemia and mortality. BMT from WT1 vaccinated MHC-matched donors including LP/J and C3H.SW, but not C57BL/6 syngeneic donors, into C57BL/6 recipient tumor-bearing mice was effective as a therapeutic maneuver and resulted in eradication of luciferase-labeled FBL3 leukemia and survival of 70–90% of mice. Interestingly, the transfer of total CD8+ T cells from immunized donors was more effective than the transfer of WT1-tetramer+CD8+ T cells, likely as a result of alloreactive and tumor-antigen reactive T cells contained with the donor total CD8+ T cells. Total and tetramer+CD8+ T cells required CD4+ T cell help for maximal anti-tumor activity, which was equivalent in efficacy from immunized or unimmunized CD4+ T cell donors. Total CD4+ T cells, alone, from immunized donors provided no anti-tumor activity. The infused donor LP/J or C3H.SW CD8+ T cells collected from cured C57BL/6 recipients, were highly reactive against WT1-expressing FBL3 leukemia cells (14% IFN-γ+) compared to non-WT1-expressing H11 leukemia cells (5% IFN-γ+). The circulating, WT1-tetramer+CD8+ T cell population expanded in cured recipients, peaking at 3.5% on day 50 and contracting through day 100 post-BMT to 0.56%. These findings show that peptide vaccination of donor mice with a tumor antigen dramatically enhances GvT activity and is synergistic with allogeneic BMT. This novel and broadly applicable approach, using leukemia-associated antigen immunization to enhance GvT by creating an “educated” donor T cell graft for allogeneic transplantation of patients with acute myeloid leukemia and myelodysplastic syndrome, is currently being translated to a Phase 1 clinical trial at our institution. Disclosures: No relevant conflicts of interest to declare.

Enhanced IFN-γ Production in Vitro by CD8+ T Cells in Hemophiliacs with AIDS as Demonstrated on the Single-Cell Level

1999 ◽  
Vol 92 (1) ◽  
pp. 111-117 ◽  
Author(s):  
Y. Pae ◽  
H. Minagawa ◽  
J. Hayashi ◽  
S. Kashiwagi ◽  
Y. Yanagi
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Single Cell Level ◽  
Cell Level ◽  
Ifn Γ

scholarly journals Changes of Host Immunity Mediated by IFN-γ+ CD8+ T Cells in Children with Adenovirus Pneumonia in Different Severity of Illness

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2384
Author(s):  
Ruilin Zheng ◽  
Yinghua Li ◽  
Danyang Chen ◽  
Jingyao Su ◽  
Ning Han ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Cell Model ◽  
Severity Of Illness ◽  
Host Immunity ◽  
Adenovirus Infection ◽  
Healthy Control ◽  
Adenovirus Pneumonia ◽  
Ifn Γ

The host immunity of patients with adenovirus pneumonia in different severity of illness is unclear. This study compared the routine laboratory tests and the host immunity of human adenovirus (HAdV) patients with different severity of illness. A co-cultured cell model in vitro was established to verify the T cell response in vitro. Among 140 patients with confirmed HAdV of varying severity, the number of lymphocytes in the severe patients was significantly reduced to 1.91 × 109/L compared with the healthy control (3.92 × 109/L) and the mild patients (4.27 × 109/L). The levels of IL-6, IL-10, and IFN-γ in patients with adenovirus pneumonia were significantly elevated with the severity of the disease. Compared with the healthy control (20.82%) and the stable patients (33.96%), the percentage of CD8+ T cells that produced IFN-γ increased to 56.27% in the progressing patients. Adenovirus infection increased the percentage of CD8+ T and CD4+ T cells that produce IFN-γ in the co-culture system. The hyperfunction of IFN-γ+ CD8+ T cells might be related to the severity of adenovirus infection. The in vitro co-culture cell model could also provide a usable cellular model for subsequent experiments.

Double Loading of Dendritic Cell MHC Class I and MHC Class II with an AML Antigen Repertoire Enhances Primary and Secondary T-Cell Responses In Vitro.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2529-2529
Author(s):  
William K. Decker ◽  
Dongxia Xing ◽  
Sufang Li ◽  
Simon N. Robinson ◽  
Hong Yang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Cd8 T Cells ◽  
Class Ii ◽  
T Cell Response ◽  
Class I ◽  
Cross Presentation ◽  
Ifn Γ

Abstract Despite improvements in therapy for acute myelogenous leukemia (AML), a significant percentage of patients still relapse and succumb to their disease. Dendritic cell immunotherapy offers the promise of potentially effective supportive therapy for a variety of neoplastic conditions; and the use of DCs loaded with tumor antigens is now recognized as an important investigational therapy. Though a variety of methods have been used to load DC vaccines, the loading of the MHC class II compartment with tumor lysate has predominated. The priming of a class II-mediated (CD4) T-cell response may be crucial to the success of DC immunotherapy as such a response is likely required for the development of memory CD8+ T-cells. DC cross-presentation is credited with the ability of lysate-loaded DCs to prime both CD4 and CD8 T-cell responses, enabling the generation of CD8+ CTLs without the loading of the MHC class I compartment (i.e. the cytoplasm). Recently, however, several reports have raised doubts as to the efficiency of cross-presentation as a mechanism for CTL priming in vivo. To examine this issue, we have loaded human DCs with both AML tumor lysate and mRNA. This technique allows the full repertoire of class I antigens to be presented without dependence upon cross-presentation; and, moreover, provides a full complement of class II antigens necessary for CD4 T-cell priming and the generation of memory responses. Methods: CD14+ precursors were isolated from normal donor PBPCs by magnetic separation. Immature DCs were then generated by culturing precursors for six days in GM-CSF and IL-4. Lysate was produced by three successive freeze/thaw cycles of blasts. mRNA was extracted from blasts using Trizol and oligo-dT separation. Immature DCs were pulsed for three hours with AML lysate and subsequently electroporated with AML mRNA. Loaded DCs were matured for 48 hours with IL-1β, TNF-α, IL-6, and PGE2 and then used to prime autologous T-cells. Short-term responses were assayed on day 5 of the 1st stimulation. Memory responses were assayed on day 10 of a tertiary stimulation. Results: Doubly-loaded DCs can prime a superior T-cell response in vitro in comparison to that of singly-loaded DCs, demonstrating a 30–70% increase in IFN-γ ELISpots over lysate-loaded DCs (p&lt;0.001) and a 3–4 fold increase in ELISpots in comparison to mRNA loaded DCs (p&lt;0.001). These results were verified by flow cytometry which showed 35% of CD8+ T-cells primed by doubly-loaded DCs were CD69+/IFN-γ+ vs. 14% of CD8+ T-cells primed by lysate-loaded DCs (p&lt;0.001). This enhancement may be based upon both an upregulation of CD83 surface expression (p&lt;0.0019) of doubly-loaded DCs and/or the upregulation of B7.1/B7.2 that accompanies elevated CD40L signaling. Memory responses were also greatly improved, with a 126% increase in total ELISpots (double loaded DCs versus lysate loaded DCs; p&lt;0.03) and a 187% increase in total IFN-γ secretion (p&lt;0.03). Unloaded (p&lt;0.01) and mRNA (p&lt;0.007) loaded DCs exhibited a virtual inability to generate memory T-cells in vitro, suggesting that the perpetuation of the memory response is reliant upon T-cell help. Conclusion: DCs doubly-loaded with lysate and mRNA are more efficient in the generation of primary and secondary immune responses than are singly-loaded DCs. The clinical administration of such doubly-loaded DCs may offer an important therapeutic option to patients with AML.

Cytomegalovirus pp65 - Recombinant Protein: A New Antigen for Efficient Restimulation of CD4+ and CD8+ pp65-Specific T Lymphocytes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3163-3163
Author(s):  
Anne Richter ◽  
Patricia Marschall ◽  
Marie Mohn ◽  
Uwe Odenthal ◽  
Silke Gösling ◽  
...  
Keyword(s):  
T Cells ◽  
Recombinant Protein ◽  
Cd8 T Cells ◽  
Matrix Protein ◽  
Peptide Pool ◽  
Effector Memory ◽  
Protein Antigens ◽  
Short Term ◽  
Ifn Γ

Abstract Short-term restimulation assays combined with the analysis of effector function, in particular the detection of cytokine production, are useful tools for the analysis and isolation of antigen-specific T cells. Until now, restimulations with soluble protein antigens failed to efficiently reactivate CD8+ T cells. We have developed a recombinant protein of the immunodominant cytomegalovirus (CMV) matrix protein pp65 for in vitro restimulation of pp65-specific CD4+ as well as CD8+ T cells. The efficiency of the CMV pp65 - Recombinant Protein to reactivate pp65-experienced CD4+ and CD8+ T cells and the specificity of the restimulated T cells were analysed. PBMC from CMV seropositive donors were restimulated with CMV pp65 - Recombinant Protein or a complete pool of overlapping pp65 peptides. Afterwards T cells were analysed for intracellular IFN-γ production by flow cytometry. Interestingly, we observed that stimulation with CMV pp65 - Recombinant Protein results in IFN-γ production in CD4+ as well as CD8+ T cells with frequencies comparable to that using the peptide pool as antigen (n=17). In contrast, upon stimulation of PBMC from CMV seronegative donors with CMV pp65 - Recombinant Protein neither IFN-γ nor TNF-α were detectable in T cells (n=6). Furthermore, we tested the specificity of CMV pp65 - Recombinant Protein-reactive CD4+ and CD8+ T cells. Therefore, IFN-γ-producing T cells were magnetically isolated after short-term stimulation with pp65 using the IFN-γ cytokine secretion assay and expanded for 7 days. Subsequently, the isolated and expanded CD4+ and CD8+ T cells were restimulated with pp65 peptide pool. More than 80 % of the CD4+ and CD8+ T cells produced IFN-γ and more than 80 % of the CD8+ T cells were positively stained with MHC class I/pp65 tetramers. These results demonstrate that CMV pp65 - Recombinant Protein efficiently and specifically reactivates pp65-experienced CD4+ as well as CD8+ T cells. Therefore, CMV pp65 - Recombinant Protein is a useful antigen for the detection and isolation of pp65-experienced CD4+ and CD8+ effector/memory T cells.

Cytomegalovirus (CMV)-Specific CD8+ T Cells Are Associated with Control of Viral Reactivation Post-Hemopoietic Stem Cell Transplantation (HSCT) Only If Shown to Be Functionally Active.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2213-2213
Author(s):  
Stephanie Verfuerth ◽  
Karl S Peggs ◽  
Arnold Pizzey ◽  
Noha Chowdhry ◽  
Stephen Mackinnon
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
The Other ◽  
Viral Reactivation ◽  
Cd8 Cells ◽  
Ifn Γ ◽  
Antiviral Chemotherapy ◽  
Cmv Disease ◽  
Cmv Reactivation

Abstract Targeted antiviral chemotherapy has greatly reduced the incidence of CMV disease post-HSCT, but adverse side effects still make CMV seropositivity a negative prognostic factor for survival. Immune protection from CMV disease depends on the adaptive immune response, and various strategies for adoptive cellular therapy for CMV have been evaluated in clinical trials to reduce the dependency on antiviral drugs. Characterizing the development of post-transplantation CMV-specific T cell responses may help identify minimum requirements for successful immunologic control of CMV reactivation. We investigated CMV-specific T cell immune reconstitution in 15 patients who all received ACT for CMV 4 weeks post-HSCT. The CMV seropositive donor-derived ACT product consisted of 1x105 CD4+ and CD8+ T cells/kg, generated through 2-week in vitro co-culture with CMV antigen-pulsed autologous dendritic cells. No graft versus host disease &gt;grade II occurred in the study group. CMV-specific T cells were characterized at various time points before, during, and after CMV reactivation events by IFN-γ secretion assay, and/or by phenotyping with HLA CMV pp65 and/or IE-1 peptide pentamers. Three patients who remained CMV PCR negative throughout had no detectable levels of CMV-specific T cells, whereas high levels of CMV-specific T cells developed in the other 12 patients who all experienced episodes of viral reactivation. 11/12 of the patients with CMV reactivation had pre-emptive antiviral chemotherapy, 1/12 controlled CMV reactivation unaided. 3/12 patients experienced a second episode of viral reactivation, which they controlled unaided. At the time of first CMV DNA detection, 1 week before the start of pre-emptive chemotherapy, pentamer+ cells were already detectable in 3/5 patients tested, with absolute numbers ranging from 1150-4900/ml blood. Only after a further 1 to 4 more weeks did T cells secreting IFN-γ in response to in vitro restimulation with CMV peptides first appear in 5 patients tested. By the time viral DNA had become undetectable post-antiviral chemotherapy or post-immunological control of viral replication, pentamer+ cells were present in 8/8 patients, with absolute numbers of pentamer+ T cells &gt;10000/ml in 7/8 patients, a level often quoted as ‘protective’ (median: 37530 cells/ml, range: 3200–139300/ml). CMV-specific CD8 T cells were maintained at high levels long after resolution of CMV viraemia (median: 24200/ml, range: 5100–75900). Absolute numbers of IFN-γ + T cells also reached high levels shortly before to just after the end of a CMV reactivation episode in 5/5 patients tested (CD8+IFN-γ + median: 48490/ml, range: 2150–138290), but subsequently temporarily dropped again to undetectable levels in some patients. Four patients had CMV reactivation episodes that were immunologically controlled unaided. Just prior these four reactivation events there was a total lack of CD8+ T cells functionally responsive to CMV. This was in spite of the presence CMV-specific CD8+ cells by phenotype in 3/3 patients tested, at &gt;10000/ml in one patient. 0% of these pentamer+ cells secreted IFN-γ in response to CMV peptide in vitro. Soon after, by the time of the first CMV PCR+ result CD8+ IFN-γ + T cells were already detected in all patients tested (median: 2180 cells/ml, range: 550–48480, n=3), with up to 64% of pent+ cells secreting IFN-γ. Pent+ cells were above the 10000 threshold in 2/3 patients tested at this time point. These data show that characterizing T cells by phenotype alone does not permit conclusions about their functionality, because some patients experienced CMV reactivation events in the presence of high levels of pentamer+ cells. Viral reactivation episodes however appeared to be associated with a prior lack of CMV-specific CD8+ cells capable of IFN-γ secretion. Immunological control of reactivation, on the other hand, may be associated with the ability to make rapid CMV-induced IFN-γ responses, because only patients with self-resolving CMV infection developed CD8+IFN-γ + responses immediately at the onset of viraemia. However it is not possible to know how many of the patients who received antiviral chemotherapy would have been able to control the infection unaided.

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