scholarly journals Tetramer-based quantification of cytomegalovirus (CMV)–specific CD8+ T lymphocytes in T-cell–depleted stem cell grafts and after transplantation may identify patients at risk for progressive CMV infection

Blood ◽  
2001 ◽  
Vol 98 (5) ◽  
pp. 1358-1364 ◽  
Author(s):  
Jan W. Gratama ◽  
Joost W. J. van Esser ◽  
Cor H. J. Lamers ◽  
Claire Tournay ◽  
Bob Löwenberg ◽  
...  
Keyword(s):  
At Risk ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Mediated Immunity ◽  
Hematopoietic Stem ◽  
Sensitive Tool ◽  
Patients At Risk ◽  
Cmv Disease

Recovery of cytomegalovirus (CMV)–specific T-cell–mediated immunity after allogeneic hematopoietic stem cell transplantation (SCT) is critical for protection against CMV disease. The study used fluorochrome-conjugated tetrameric complexes of HLA-A2 molecules loaded with the immunodominant NLVPMVATV (NLV) peptide derived from the CMV protein pp65 to quantify A2-NLV–specific CD8+ T cells in partially T-cell–depleted grafts administered to 27 HLA-A*0201+ patients and to monitor recovery of these T cells during the first 12 months after SCT. None of the 9 CMV-seronegative patients became infected with CMV, whereas 14 of 18 CMV-seropositive patients developed CMV antigenemia after SCT. CMV-seropositive recipients of grafts from CMV-seronegative donors required more preemptive treatment with ganciclovir (GCV) than those of grafts from CMV-seropositive donors (3 [1-6] versus 1 [0-3] courses, respectively; P = .009). The number of A2-NLV–specific CD8+ T cells in the grafts correlated inversely with the number of preemptive GCV courses administered (r = −0.61; P = .01). None of the 9 CMV-seronegative patients mounted a CMV-specific immune response as measured by monitoring A2-NLV–specific CD8+ T cells after SCT. Thirteen of 14 CMV-seropositive patients without CMV disease recovered these T cells. In spite of preemptive GCV treatment, CMV disease developed in 4 patients, who all failed to recover A2-NLV–specific CD8+ T cells after SCT(P = .002). Thus, enumeration of HLA-restricted, CMV-specific CD8+ T cells in the grafts and monitoring of these T cells after SCT may constitute a rapid and sensitive tool to identify SCT recipients at risk for developing CMV disease.

Functional Comparison and Longitudinal Assessment of Tri-Functional T-Cells Recognizing CMV pp65 and IE-1 Polypeptides in Hematopoietic Stem Cell and Solid Organ Transplant Recipients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2936-2936
Author(s):  
Don J. Diamond ◽  
Simon F. Lacey ◽  
Corinna La Rosa ◽  
Wendy Zhou ◽  
Ghislaine Gallez-Hawkins ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Solid Organ ◽  
Transplant Recipients ◽  
Hematopoietic Stem ◽  
Specific Ctls ◽  
Tnf Α ◽  
Ifn Γ ◽  
Cmv Disease

Abstract Reconstitution of adaptive T-cell responses to human cytomegalovirus (CMV) is critical to protection from CMV disease following hematopoietic stem cell (HSCT) or solid organ transplantation (SOT). However, there is an incomplete understanding of which CMV antigens and epitopes are most crucial to providing protective responses. The functional status of cytotoxic T-lymphocyte (CTL) populations recognizing cytomegalovirus IE-1 and pp65 polypeptides was investigated in PBMC from either HSCT or SOT recipients. Our previous finding of differing levels of degranulation between CMV IE1 and pp65/pp50 specific T-cells was complicated by the possibility that differences were epitope and/or HLA-specific. We generalized the approach using a combined flow-based CD107a/b degranulation/mobilization and intracellular cytokine (ICC) assays using peptide libraries as antigens. These assays indicated that a significantly higher proportion of pp65-specific CTLs were in a more mature functional state compared to IE-1-specific CTLs. Degranulation/multicytokine ICC assays also indicated that a significantly higher proportion of the pp65-specific versus IE-1-specific CTLs secreted both IFN-γ and TNF-α, in addition to possessing greater cytotoxic potential. These results support our earlier findings of functional differences between CTLs recognizing individual epitopes within the IE-1 and pp65 antigens in HSCT recipients, and extend them to a broader array of HLA-restricted responses to those antigens. A report that a subset of HIV-1 specific CTLs capable of producing both IFN-γ and TNF-α was associated with improved cytotoxic activity prompted us to investigate whether degranulation, a functional correlate of cytotoxicity, was positively associated with dual cytokine production and predicted differences between IE1 and pp65-specific CD8+ T-cells. A higher proportion of pp65-specific compared to IE1-specific T-cells were present in the trifunctional IFN-γ+,TNF-α+, CD107+ population (p=0.008) in HSCT recipients. We have extended these findings to investigate the role of donor CMV status in terms of functional maturity of CMV-specific T cell response in transplant recipients. T cell maturation/function may act as a mechanistic correlate to the survival advantage of recipients receiving a stem-cell graft from CMV sero-positive donors. These principles have also been applied to investigations of a high risk population of sero-negative recipients of a sero-positive liver allograft. Data from this study will also be reviewed in the context of the model of trifunctional T cells being indicative of enhanced protective capacity against CMV disease and associated with survival.

New Interleukin-15 Superagonist (IL-15 SA) Significantly Enhances Graft-Versus-Tumor Activity After Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3254-3254
Author(s):  
Cavan P Bailey ◽  
Christopher Sauter ◽  
Michelle M Panis ◽  
Tulin Budak-Alpdogan ◽  
Hing Wong ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Cd8 T Cells ◽  
T Cell Proliferation ◽  
Hematopoietic Stem ◽  
Interleukin 15 ◽  
And Function

Abstract Interleukin-15 (IL-15) is a pleiotropic cytokine, which plays various roles in the innate and adaptive immune system, including the development, activation, homing and survival of immune effector cells. IL-15 has been previously shown to increase CD8+ T and NK cells number and function in normal mice and recipients of stem cell transplantation. However, obstacles remain in using IL-15 therapeutically, specifically its low potency and short in vivo half-life. To overcome this, a new IL-15 mutant (IL-15N72D, J. Immunol, 2009; 183:3598) has been developed, with increased biological activity. Co-expressing IL-15N72D, in conjunction with IL-15RαSu/Fc produced a biologically active and highly potent IL-15 superagonist complex (IL-15SA, also known as ALT-803, Cytokine, 2011; 56:804). We evaluated the effects of IL-15-SA on immune reconstitution and graft-versus-tumor (GVT) activity in recipients of allogeneic hematopoietic stem cell transplantation (HSCT). Lethally irradiated BALB/c recipients were transplanted with T-cell depleted (TCD) bone marrow (BM) cells from B6 mice. IL-15 SA was administered via IP injection in two doses on days +17 and +24 after transplant. Animals were sacrificed at day 28. Administration of IL-15 significantly increased the numbers of CD8+ T cells and NK cells. IL-15 SA also augmented interferon-γ secretion from CD8+ T cells. We observed similar activity in B6CBA→CB6F1 transplant model. Interestingly IL-15 SA upregulates NKG2D and CD107a expression on CD8+ T cells. IL-15 SA administration also specifically increased slow-proliferative CD8+ T-cell proliferation in conjunction with robust IFN-γ and TNF-α secretion in CD8+ T cells in recipients of CFSE (carboxyfluorescein succinimidyl ester) labeled-T-cell infusion, whereas there was no effect on CD4+ T-cell proliferation. We then tested the anti-tumor activity of IL-15 SA in three different tumor models; murine mastocytoma (P815), murine B cell lymphoma (A20) and murine renal cell carcinoma (Renca). We found that IL-15 SA administration enhanced GVT activity against P815 and A20 in recipients of allogeneic HSCT though this activity required a low-dose T cell infusion with HSCT. Interestingly, augmented GVT activity against to Renca after IL-15 SA administration in recipients of allogeneic HSCT did not require T cell infusion. We conclude that IL-15 SA is a very potent cytokine complex for enhancing CD8+ and NK cell reconstitution and function after HSCT, which would be a candidate for post-transplant immunotherapy. Disclosures: Wong: Altor Bioscience: Employment.

scholarly journals Combinatorial Cytokine Secretion Signature of Donor-Derived T Cells Infused with the Graft: A New Potential Biomarker of Acute Graft-Versus-Host Disease in Αβt-Cell/CD19 B-Cell Depleted Hematopoietic Stem Cell Transplant Recipients

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1684-1684
Author(s):  
Raul Montiel-Esparza ◽  
Giulia Barbarito ◽  
Samantha Peck ◽  
Magali Bazzano ◽  
Rachana Patil ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Single Cell ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Cytokine Secretion ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Tnf Α

Abstract Background: Hematopoietic stem cell graft manipulation strategies, such as αβT-cell/CD19 B-cell depleted hematopoietic stem cell transplantation (αβhaplo-HSCT), address the lack of matched donors and reduce the incidence of severe acute graft-versus-host disease (aGvHD). However, grade II-IV aGvHD still occurs in 25-30% of αβhaplo-HSCT recipients . Studies aimed at understanding the pathogenesis underlying aGVHD in αβhaplo-HSCT are lacking. We hypothesized that αβT cells adoptively transferred with the HSCT (<1x10 5/Kg) have unique combinatorial cytokine secretion signatures that may predict the occurrence of aGvHD. Here we used the IsoPlexis single-cell proteomics for CD4 + and CD8 + T cells to identify those putative signatures . Methods: Six patients with hematologic malignancies receiving fully myeloablative αβhaplo-HSCT at Lucile Packard Children's Hospital, Stanford, between 08/2018 and 05/2020 were enrolled upon signing IRB approved informed consent. Three patients developed grade II-IV aGvHD, while three did not. Aliquot of the graft and of peripheral blood collected at the time of aGvHD onset or at corresponding time points for the patients who did not develop aGvHD, were analyzed. Single sorted CD4 + and CD8 + T cells were profiled by single-cell barcode chip assay from IsoPlexis system (IsoPlexis, Branford, CT) after stimulation with PMA (50 ng/mL) and Ionomycin (1mcg/mL). Following the Human Adaptive Immune Panel, cytokines from CD4 + and CD8 + single T cells were captured by fluorescence ELISA, which measured the numbers of cytokine-producing cells (secretion frequency) and numbers of cytokines produced by individual cells across five functional groups: effector, stimulatory, chemoattractive, regulatory and inflammatory (Table 1). Polyfunctionality was defined as the secretion of 2+ cytokines from each CD4 + and CD8 + T cell. The T cell polyfunctional strength Index (PSI) was defined as the percentage of polyfunctional cells, multiplied by the sum of the mean fluorescence intensity of the proteins secreted by those cells. Additional statistical analysis was performed using the Student's t test. Results: We compared the combinatorial cytokine secretion signature of individual CD4 + and CD8 + T cells isolated from grafts infused into patients, who eventually did or didn't develop aGvHD. We are comparing the signature of post-HSCT CD4 + and CD8 + T cells isolated from patients who did or did not develop aGvHD. Collectively, we considered three variables: cytokine secretion frequency, numbers of cytokines produced by individual cells and characteristics of the cytokines secreted (functional group) upon stimulation. Single-cell functional heterogeneity evaluated by t-Distributed Stochastic Neighbor Embedding (t-SNE), showed higher CD4 + and CD8 + T-cell polyfunctionality (up to 4+ cytokines) with effector and stimulatory dominant functions in the grafts of patients who developed aGvHD, compared to those who did not develop aGvHD (Fig1). The average PSI (driven by Granzyme B, TNF-α, IFN-γ, MIP-1β, IL2, and IL-8) was found to be higher in both CD4 + and CD8 + T cells from the grafts of patients who developed aGvHD (Fig 2). Combinatorial cytokine secretion analysis showed that T cells from grafts of patients who did not develop aGvHD had unique signatures with CD4 + T cells having the predominant cytokine secretion signature of IL2 and TNF-α, and CD8 + T cells having three predominant cytokine secretion signatures: IL2, IL8, TNF-α; MIP-1β, IL8; and MIP-1β, IFN-γ (Fig3). Conclusions: Preliminary data from αβhaplo-HSCT pediatric recipients obtained using IsoPlexis single-cell functional proteomics for CD4 + and CD8 + T cells showed that an increased donor T-cell polyfunctionality with a Th1 dominant functional phenotype may be predictive of an increased risk of aGvHD, while CD4 + and CD8 + T cells infused into patients who didn't develop aGvHD, had combinations with limited cytokine secretion signatures. Ongoing analysis suggest that polyfunctional CD8 + T cells present in the graft of patients who developed aGvHD, are present at the time of aGvHD initiation, while the polyfunctional CD4 + T cell are not present at the onset of aGvHD. Correlation with ongoing studies on circulating cytokines and clonotypic analysis of αβT cells infused with the graft will be crucial to elucidate the cross talking between the donor's immune system and recipient's inflammatory milieu. Figure 1 Figure 1. Disclosures Parkman: Jasper Biotech: Consultancy. Bertaina: Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; AdicetBio: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Immunosenescent Characteristics of T Cells in Young Patients Following Haploidentical Stem Cell Transplantation from Parental Donors

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3283-3283
Author(s):  
Ga Hye Lee ◽  
Kyung Taek Hong ◽  
Jung Yoon Choi ◽  
Hee Young Shin ◽  
Won-Woo Lee ◽  
...  
Keyword(s):  
Dna Damage ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Telomere Length ◽  
Cell Transplantation ◽  
Cd8 T Cells ◽  
Hematopoietic Stem

Introduction: Pediatric and adolescent patients in need of allogeneic hematopoietic stem cell transplantation generally receive stem cells from older, unrelated or parental donors when a sibling donor is not available. Despite encouraging clinical outcomes, it has been suggested that immune reconstitution accompanied by increased replicative stress and a large difference between donor and recipient age may worsen immunosenescence in pediatric recipients. Therefore, in this study paired samples were collected at the same time from donors and recipients of haploidentical hematopoietic stem cell transplantation (HaploSCT). Methods: We conducted flow cytometry-based phenotypic and functional analyses and telomere length measurements of 21 paired T-cell sets from parental donors and children who received T cell-replete HaploSCT with post-transplant cyclophosphamide (PTCy) at Seoul National University Children's Hospital between February 2014 and January 2017. The conditioning regimen was comprised of targeted busulfan (total target area under the curve, 75,000 mg•h/L) with intensive pharmacokinetic monitoring, fludarabine and cyclophosphamide. Results: Fourteen pediatric, adolescent, and young adult patients with malignant disease and seven with nonmalignant disease were included with a median post-transplantation period of 16.9 months (range, 12.4-38.8). Senescent T cells, CD28- or CD57+ subsets of both CD4+ and CD8+ T cells, were significantly expanded in patients compared with parental donors. Further, not only CD4+CD28- T cells, but also CD4+CD28+ T cells showed reduced cytokine production capacity and impaired polyfunctionality compared with parental donors, whereas their TCR mediated proliferation capacity was comparable. Of note, the telomere length in patient T cells was preserved, or even slightly longer, in senescent T cells compared with donor cells. We also found that the patients had a higher level of γ-H2AX-expressing CD28- senescent T cells compared with the donors, which is used as a DNA damage marker. Regression analysis showed that senescent features of CD4+ and CD8+ T cells in patients were influenced by donor age and the frequency of CD28- cells, respectively. Conclusions: Our data suggest that T cells undergo premature immunosenescent changes and exhibit functional defects in pediatric HaploSCT recipients. Further, there is an increased level of DNA damage in patient CD4+ T cells compared to those of parental donors. Therefore, long-term, comprehensive immune monitoring of these patients is necessary. Disclosures No relevant conflicts of interest to declare.

Development of Novel Stem Cell Transplantation and Gene-Immunotherapy Using WT1-Specific T-Cell Receptor Gene.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3028-3028
Author(s):  
Toshiki Ochi ◽  
Hiroshi Fujiwara ◽  
Kozo Nagai ◽  
Toshiaki Shirakata ◽  
Kiyotaka Kuzushima ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Hematopoietic Stem Cells ◽  
Cell Transplantation ◽  
Clinical Efficacy ◽  
Cd8 T Cells ◽  
Hematopoietic Stem

Abstract Abstract 3028 Poster Board II-1004 Purpose The Wilms' tumor 1 (WT1) is one of the zinc-finger transcriptional regulators, and its expression level is very low in most tissues of adults. In contrast, various kinds of leukemia and solid tumors express WT1 abundantly, and high expression level of WT1 is correlated with disease aggressiveness and poor prognosis. These findings indicate that WT1 is a promising target antigen for anti-cancer cellular immunotherapy. Following identification of immunogenic epitopes derived from WT1 which are recognized by HLA class I-restricted and HLA class II-restricted T cells, phase I/II WT1 peptide vaccination trials have been conducted. Although the positive correlation between the clinical efficacy and vaccine-induced WT1-specific T-cell response has been reported, the clinical efficacy is not satisfactory. Adoptive transfer of WT1-specific T cells seems to be the promising approach to achieve marked improvement in clinical efficacy of WT1-targeting immunotherapy, however, it still remains difficult to expand WT1-specific T cells sufficiently ex vivo. To overcome these problems, we attempted to establish gene-immunotherapy targeting WT1 using T-cell receptor (TCR) gene isolated from the WT1-specific T-cell clone. We also verified the feasibility of novel stem cell transplantation by transducing WT1-specific TCR gene into hematopoietic stem cells. Methods We cloned the full length TCR-αa and -β genes from a WT1235-243-specific and HLA-A*2402-restricted cytotoxic T lymphocyte (CTL) clone. The WT1-specific TCR gene-repressing retroviral and lentiviral vectors were constructed. Retroviral vector was transduced to human peripheral T cells in retronectin-coated plate. WT1-specific functions of TCR gene-transduced CD8+ T cells and CD4+ T cells were examined by evaluating WT1 peptide-specific cytotoxicity by 51Cr-release assay and WT1 peptide-specific Th1 cytokine production, respectively. To improve the efficacy of WT1-specific TCR expression, we developed the novel retroviral vector which can inhibit selectively intrinsic TCR expression (si-TCR vector). Finally, we transduced the WT1-specific TCR lentiviral vector into human cord blood CD34+ cells, and transplanted them to NOD/SCID/common-γnull mice. Then, we examined whether WT1-specific human mature T cells can differentiate in mice. The presence of WT1-specific human T cells in mice was determined by tetramer assay and IFN-γ production in response to stimulation with WT1 peptide. Results Following transfer of WT1-specific TCR gene into peripheral blood lymphocytes, WT1 peptide-specific CD8+ and CD4+ T cells could be expanded easily in vitro. TCR gene-transduced CD8+ T cells exerted cytotoxicity against WT1 peptide-pulsed target cells and human leukemia cells in an HLA-A*2402-restricted manner. Similarly, TCR gene-transduced CD4+ T cells showed WT1-specific Th1 cytokine production in response to stimulation with human leukemia cells in HLA-A*2402-restricted fashion depending on the interaction of CD4 and HLA class II molecules. The newly developed si-TCR vector appeared to inhibit expression of endogenous TCR efficiently and improved the efficacy of WT1-specific TCR expression 3 to 5-fold higher as compared to the conventional vector. Three months after transplantation of WT1-specific TCR gene-transduced human hematopoietic stem cells in NOD/SCID/common-γnull mice, differentiation of WT1-specific human T cells in murine spleen was evaluated. Tetramer assay revealed that human mature T cells expressing WT1-specific TCR on their cell surface were clearly detected. Furthermore, these WT1-specific CD8+ T cells appeared to produce IFN-γ in response to stimulation with WT1 peptide-loaded HLA-A*2402-positive cells. Conclusion The adoptive gene-immunotherpay using WT1-specific TCR gene against leukemia seems to be promising. Moreover, the novel stem cell transplantation using WT1-specific TCR gene-transduced hematopoietic stem cells might open the door to induce long-lasting anti-leukemic cellular immunity in patients with leukemia. Disclosures No relevant conflicts of interest to declare.

Long-Term Persistence of Functionally Altered GPI-Anchor Negative T-Cell Subsets After Alemtuzumab-Based T-Cell Depleted Hematopoietic Stem Cell Transplantation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2438-2438
Author(s):  
Eva M Wagner ◽  
Aline N Lay ◽  
Timo Schmitt ◽  
Julia Hemmerling ◽  
Diana Wolff ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Cd8 T Cells ◽  
T Cell Subsets ◽  
Gpi Anchor ◽  
Ifn Gamma ◽  
Hematopoietic Stem

Abstract Abstract 2438 Poster Board II-415 The anti CD52 antibody alemtuzumab is frequently used for in vivo T cell depletion (TCD) in the context of allogeneic hematopoietic stem cell transplantation (HSCT). We have recently demonstrated the persistence of CD52-negative T-cell subsets in patients after HSCT following alemtuzumab-mediated TCD (Meyer, Wagner et al., Bone Marrow Transplantation 2009). The loss of CD52 among lymphocytes was exclusively related to T cells and was more prominent in CD4 compared to CD8 T cells. CD8-depleted donor-lymphocyte infusions (DLI) increased the percentage of CD52-positive CD4 T cells. In patients who did not receive DLI, CD52-negative T cells were detected in significant proportions of up to 40% found even more than 3 years after transplantation. We therefore investigated the regulation as well as the functional consequences of a loss of CD52-expression in T cells of our patients. Peripheral blood T cells of patients with CD52-negative T cells after more than 12 months post allogeneic HSCT following TCD with high-dose alemtuzumab (100 mg) were sorted according to their expression of CD52. RT-PCR showed no difference in CD52 mRNA expression of CD52-positive compared to negative T cells. Since transcriptional regulation was therefore unlikely and CD52 is a glycosylphosphatidylinositol (GPI)-anchored protein, we stained for the presence of further GPI-anchored molecules such as CD55 and CD59 on peripheral blood lymphocytes of our patients. We found that the CD52-negative T cells had also lost expression of CD55 and CD59, whereas CD52-positive cells remained positive for these antigens. We then directly labeled the GPI-anchors using FLAER (fluorescent aerolysin) and thereby confirmed that the loss of CD52 was correlated with a reduced density of the GPI-anchors in the cell-membrane. However, our patients did not exhibit clinical signs of paroxysmal nocturnal hemoglobinuria (PNH), which is in line with the finding that the loss of GPI-anchors was only related to T cells. With the aim to characterize the functional impact of the reduced GPI-anchor density on T cells, we separated CD52-negative from CD52-positive T cells by flow cytometry. The subpopulations were expanded in vitro using low-dose IL2, OKT3, and allogeneic feeder-cells. CD52 expression remained unaltered in CD52-negative as well as CD52-positive cultures for more than 6 weeks. In contrast, when purified T cells of healthy donors were treated with alemtuzumab in vitro (10 μg/mL, 4 h), we only observed a transient down-regulation of the antigen. The growth-kinetics of the non-specifically stimulated T cell cultures did not differ between the CD52-positive and the negative cultures. Yet, when we expanded T cells of a cytomegalovirus (CMV)-positive patient, transplanted from a CMV-positive donor, by subsequent stimulation with overlapping peptides of CMV-pp65, only the proliferation of CD52-positive T cells increased after the addition of peptides. We furthermore applied CD52-positive as well as CD52-negative CD4 and CD8 T cells derived from the antigen-independent culture of this patient in an IFN-gamma ELISPOT assay with autologous dendritic cells (DC) loaded with overlapping peptides of CMV-pp65 and IE1. CMV-specific IFN-gamma spot-production was only evident in the CD52-positive populations. We also conducted IFN-gamma secretion-assays on ex vivo T cells stimulated with autologous DC loaded with CMV-peptides and found a reduced antigen-specific IFN-gamma production in CD52-negative CD4 and CD8 T cells. In addition, we analyzed IFN-gamma secretion of T cells following allogeneic stimulation with DC of a healthy individual and again detected lower levels of IFN-gamma production by CD52-negative compared to CD52-positive T cells. In summary, we demonstrated that the permanent loss of CD52 in a proportion of reconstituting T cells after alemtzumab-based TCD is associated with a loss of GPI-anchors in the cellular membrane. Our data suggest that this loss correlates with reduced T-cell effector-functions in response to antigen-specific stimulation. In addition to a better understanding of the role of alemtuzumab-mediated TCD on T cell reconstitution, further comparison of functional responses in different T-cell subsets in association with the presence or absence of GPI-anchors might help to explore the impact of GPI-anchors and GPI-anchored molecules in this context. Disclosures: No relevant conflicts of interest to declare.

Abnormalities of the Bone Marrow Immune Microenvironment in Patients with Prolonged Isolated Thrombocytopenia after Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4602-4602
Author(s):  
Yang Song ◽  
Yuan Kong ◽  
Min-Min Shi ◽  
Yu-Qian Sun ◽  
Yu Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Microenvironment ◽  
Hematopoietic Stem ◽  
Cell Subpopulations ◽  
T Cell Subpopulations

Abstract Background:Prolonged Isolated Thrombocytopenia (PT), is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and defined as the engraftment of all peripheral blood cell lines other than a PLT count ≤20×10E+9/L or dependence on PLT transfusions for more than 90 days after allo-HSCT. Nevertheless, the mechanisms underlying PT remain unclear. Recent studies have presumed that the mechanism of PT might be similar, at least in part, to that of Immune Thrombocytopenia (ITP). BM immune microenvironment is considered to be involved in the regulation of hematopoiesis, and also influence the production of platelets. There is growing evidence that activated CD8+ T cells in the bone marrow (BM) of patients with ITP might suppress megakaryocyte apoptosis, leading to impaired platelet production. In our previous study, we also found the deregulated T cells responses in BM were associated with ITP patients. Therefore, we hypothesized aberrant immune microenvironment may also influence the production of platelet after allo-HSCT, contributing to the occurrence of PT, so we conducted a study to analyze the alteration of T cell subpopulations and cytokines in BM micro-environment of allotransplant patients. Aims:To compare the cellular compositions and function of T cells in BM microenvironment between patients with PT and good graft function (GGF) after allo-HSCT. Methods:Using a prospective nested case-control study, the T cell subpopulations in BM were analyzed by flow cytometry in 15 patients with PT, 30 matched patients with GGF after allo-HSCT, and 15 healthy donors (HDs). The fractions of T cells, including Th1, Tc1,Th2, Tc2 ,Th17 and Treg were identified as CD3+CD8-IFN-gama+, CD3+CD8-IFN-gama+, CD3+CD8+IL4+, CD3+CD8+IL-4+, CD3+CD8-IL17A+ and CD3+CD4+CD25+Foxp3+, respectively. The levels of IFN-gama, IL-4 and IL-17A in BM plasma were detected by cytometric beads assay. Results: The demographic and clinical characteristics were similar between allo-HSCT patients with PT and those with GGF. The T cell subset analysis revealed that the proportion of CD8+ T cells in BM was higher in PT patients. The in vitro cytokine stimulated tests demonstrated a significant higher proportion of Th1 in PT patients (29.8% ±13.0% vs. 21.7%±12.2%, P=0.01), and we also found an elevated percentage of Tc1 in PT patients when compared with GGF (39.3% ±19.3% vs. 23.0% ± 14.0%, P=0.01). Meanwhile, the similar percentage of Th2 and Tc2 were found in PT patients. The type-1/ type-2 response ratio was calculated by the percentages of Th1/Th2 and Tc1/Tc2. A significant elevation in the ratio of Tc1/Tc2 (37.3 vs. 22.1 vs. 15.6, P<0.05) was observed in PT when compared with those in GGF and HDs, whereas the ratio of Th1/Th2 did not differ from GGF. Moreover, we also found the significant elevated percentage of Th17 (3.1% ±2.1% vs. 1.1%± 0.7%, P<0.01) and the similar percentage of Treg in PT patients compared with GGF, leading to a higher ratio of Th17/Treg (0.9 vs. 0.6 vs. 0.3, P<0.05). The changes of IFN-gama, IL-4 and IL-17A levels in BM plasma detected by cytometric beads assay were in accordance with the intracellular cytokine results analyzed by flow cytometry. Summary/Conclusion: Our study demonstrated that the abnormal BM immune microenvironment including the higher percentage of Th1, Tc1, and Th17 cells in patients with PT, suggesting that the dysfunction of T cells response in BM immune microenvironment may contribute to the occurrence of PT after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

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