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.

Prolonged Thrombocytopenia After Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) and Its Association with an Increase in CD8+ CX3CR1+ Cells in Bone Marrow.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3077-3077
Author(s):  
Xiao-hui Zhang ◽  
Guo-xiang Wang ◽  
Yan-rong Liu ◽  
Lan-Ping Xu ◽  
Kai-Yan Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Surface Expression ◽  
T Cell Subsets ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Cell Counts

Abstract Abstract 3077 Background: Since prolonged thrombocytopenia (PT) is an independent risk factor for poor clinical outcome after allogeneic hematopoietic stem cell transplantation (allo-HSCT), the underlying mechanisms need to be understood in order to develop selective treatments. Previous studies1–4 have suggested that abnormalities in B cells may play a role in the pathogenesis of PT. However, abnormalities in B cells alone do not fully explain the complete pathogenic mechanisms of PT. Our previous studies5 showed that the frequency of megakaryocytes with a ploidy value ≤ 8N was significantly increased in patients who developed PT after allo-HSCT compared to the control group. Mechanisms concerning the megakaryocyte hypoplasia in PT after allo-HSCT are not well understood. Design and Methods: PT was defined as a platelet count ≤80 × 109/L for more than 3 months after HSCT, recovery of all other cell counts, and no apparent cause for thrombocytopenia, such as aGVHD, disease recurrence, CMV infection, or antiviral drug treatment at three months post-HSCT when all other blood cell counts had return to normal.5 We analyzed T cell subsets in bone marrow (BM) and peripheral blood (PB) from allo-HSCT recipients with and without PT (n = 23 and 17, respectively) and investigated the expression characteristics of homing receptors CX3CR1, CXCR4 and VLA-4 by flow cytometry. Futhermore, Mononuclear cells (MNCs) from PT patients and controls were cultured with and without autologous CD8+ T cells in vitro, and clarify the effect of activated CD8+ T cells on the ploidy and apoptosis of megakaryocytes in the bone marrow. Results: The results demonstrated that the percentage of CD3+ T cells in the BM was significantly higher in PT patients than the experimental controls (76.00 ± 13.04% and 57.49 ± 9.11%, respectively, P < 0.001), whereas this difference was not significant for the PB (71.01 ± 11.49% and 70.49 ± 12.89%, respectively, P = 0.911). While, some T cell subsets in the BM and PB from allo-HSCT recipients with PT were not significantly different from that of the experimental control group, such as CD8+ T cells, CD4+ T cells, CD4+ CD25bright T cells (regulatory T cells), CD44hi CD62Llo CD8+ T cells and naive T cells (CD11a+ CD45RA+). Furthermore, the surface expression of homing receptor CX3CR1 on BM T cells (64.16 ± 14.07% and 37.45 ± 19.66%, respectively, P < 0.001) and CD8+ T cells (56.25 ± 14.54% and 35.16 ± 20.81%, respectively, P = 0.036), but not in blood, were significantly increased in PT patients compared to controls. For these two groups of patients, the surface expression of CXCR4 and VLA-4 on T cells and CD8+ T cells from both BM and PB did not show significant differences. Through the study in vitro, we found that the activated CD8+ T cells in bone marrow of patients with PT might suppress apoptosis (MNC group and Co-culture group: 18.02 ± 3.60% and 13.39 ± 4.22%, P < 0.05, respectively) and Fas expression (MNC group and Co-culture group: 21.10 ± 3.93 and 15.10 ± 2.33, P <0.05, respectively) of megakaryocyte. In addition, megakaryocyte with a ploidy value ≤ 8N (MNC group: 40.03 ± 6.42% and 24.54 ± 4.31%, respectively, P < 0.05) was significantly increased in patients with PT compared to the control group. Conclusions: In conclusion, an increased surface expression of CX3CR1 on T cells may mediate the recruitment of CD8+ T cells into the bone marrow in patients with PT who received an allo-HSCT. Moreover, CD8+CX3CR1+ T cells, which can have significantly increased numbers in bone marrow of patients with PT, likely caused a reduction in the megakaryocyte ploidy, and suppressed megakaryocyte apoptosis via CD8+ T cell-mediated cytotoxic effect, possibly leading to impaired platelet production. Therefore, treatment targeting CX3CR1 should be considered as a reasonable therapeutic strategy for PT following allo-HSCT. Disclosures: No relevant conflicts of interest to declare.

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.

Adoptive Transfer of In Vitro Generated T Cell Precursors Enhances Donor T Cell Reconstitution and Graft-Versus-Tumor Activity in Allogeneic Hematopoietic Stem Cell Transplantation Recipients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 63-63 ◽  
Author(s):  
Johannes L. Zakrzewski ◽  
Adam A. Kochman ◽  
Sidney X. Lu ◽  
Theis H. Terwey ◽  
Theo D. Kim ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
Growth Factors ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Hematopoietic Stem ◽  
T Cell Reconstitution

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) is associated with a varying period of immunoincompetence that particularly affects he T cell lineage resulting in significant morbidity and mortality from opportunistic infections. Recent studies have shown that murine T cells and their precursors can be generated from hematopoietic stem cells (HSC) in vitro using a OP9-DL1 coculture system consisting of OP9 bone marrow stromal cells expressing the Notch 1 ligand Delta-like 1 and growth factors (interleukin 7 and fms-like tyrosine kinase-3 ligand). In this study we determined the effects of adoptively transferred in vitro generated T cell precursors on T cell reconstitution after allogeneic HSCT. We selected HSC (Lin- Sca-1hi c-kithi) from bone marrow (BM) of C57BL/6 mice and cultured these cells on a monolayer of OP9-DL1 cells in the presence of growth factors. These HSC expanded 2,000–5,000-fold within 3–4 weeks and consisted of &gt;95% CD4-CD8-double negative (DN) T cell precursors after 16–28 days of culture. We infused these cells (8x106) with T cell depleted (TCD) BM (5x106) or purified HSC into allogeneic recipients using minor antigen mismatched and MHC class I/II mismatched transplant models. Control mice received TCD BM or purified HSC only. Progeny of OP9-DL1 derived T cell precursors were found in thymus and spleen increasing thymic cellularity and significantly improving thymic and splenic donor T cell chimerism. This effect was even more pronounced when purified HSC instead of whole BM were used as allograft. T cell receptor repertoire and proliferative response to foreign antigen (determined by third party MLR) of in vivo differentiated OP9-DL1 derived mature T cells were intact. Administration of in vitro generated T cell precursors did not induce graft-versus-host disease (GVHD) but mediated significant graft-versus-tumor (GVT) activity (determined by in vivo bioluminescence imaging) resulting in a subsequent significant survival benefit. This advantage was associated with better cytokine responses (IL-2, INF-g, TNF-a) in T cells originating from OP9-DL1 derived T cell precursors compared to BM donor derived T cells. We conclude that the adoptive transfer of OP9-DL1 derived T cell precursors significantly enhances post-transplant T cell reconstitution and GVT activity in the absence GVHD.

Increased Proliferation of FoxP3 Regulatory T Cells after Allogeneic Hematopoietic Stem Cell Transplantation Is Counterbalanced by Increased Susceptibility to Apoptosis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 720-720
Author(s):  
Ken-ichi Matsuoka ◽  
Corey Cutler ◽  
John Koreth ◽  
Joseph H Antin ◽  
Robert J Soiffer ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Chronic Gvhd ◽  
T Cell Subsets ◽  
Cd4 T Cell ◽  
Ki 67 ◽  
Hematopoietic Stem ◽  
Healthy Donors

Abstract CD4+FoxP3+ Regulatory T cells (Treg) play a critical role in the maintenance of tolerance after allogeneic hematopoietic stem cell transplantation (HSCT). We previously demonstrated that patients with active chronic graft-versus-host disease (cGVHD) have a reduced frequency of Treg. However, the mechanisms responsible for inadequate Treg reconstitution in patients with cGVHD have not been characterized. We therefore examined phenotypic and functional characteristics of Treg in 16 patients 2–41 months (median 10 months) post-HSCT to elucidate these mechanisms. Treg were compared to conventional CD4+FoxP3-T cells (Tcon) within individual patient samples and to healthy donors. All patients received TBI-based myeloablative conditioning, peripheral blood stem cells from HLA-matched donors (12 MRD; 4 URD) and acute GVHD prophylaxis (11 tacrolimus and sirolimus; 5 tacrolimus and methotrexate). At the time of analysis, 9 patients had no chronic GVHD, 5 had active chronic GVHD (1 limited disease; 4 extensive disease) and 2 had inactive chronic GVHD. Total CD4 counts were relatively low after HSCT compared to healthy donors (median CD4 273/ul vs 756/ul). After HSCT, patient Treg exhibited a predominant CD45RA(−)CCR7(−) effector/memory phenotype. Expression of CD31 on CD45RA+ Tcon and Treg was used to identify cells within these subsets that were recent thymic emigrants (RTE). In patient samples, 16.5% of Tcon and 2.8% of Treg expressed CD31+CD45RA+. In healthy donors, 22.9% of Tcon and 5.4% of Treg were CD31+CD45RA+. The lower fraction of RTE within the Treg population after transplant suggests that Treg primarily reconstitute through peripheral proliferation rather than through thymic generation. The proliferative capacity of both Tcon and Treg was examined by evaluating expression of Ki-67 in these subsets. After transplant, Ki-67 expression was significantly higher in Treg (5.2%) than in Tcon (1.5%) (p<0.001). This was significantly higher in both populations compared to healthy donors where 2.5% of Treg (p<0.05) and 0.2% of Tcon (p<0.01) expressed Ki-67. In both patients and healthy donors, Ki-67 expression was found almost entirely in cells that were CD45RA-indicating that proliferation was primarily occurring within the memory subsets of Tcon and Treg. Increased expression of Ki-67 on Treg was associated with low CD4 T cell counts (p<0.001), but not with time after HSCT (p=0.21) and chronic GVHD status (p=0.35). Treg Ki-67 expression after HSCT showed a strong positive correlation with CD95 (FAS) expression (p<0.01), but this association was not present in Tcon post-HSCT or in Treg from healthy donors. To determine whether increased expression of CD95 results in apoptosis of Treg, we purified 4 different CD4+ T cell subsets by cell sorting (CD45RA+ Tcon, CD45RA− Tcon, CD45RA+ Treg and CD45RA− Treg) from healthy donors and HSCT patients. Purified cells were cultured with or without agonistic FAS antibody (anti-FAS) and apoptosis was measured using Annexin-V staining. Anti-FAS rapidly induced apoptosis of CD45RA− memory-like Treg from HSCT patients while all other Treg and Tcon subsets were relatively resistant to apoptosis. In summary, these results indicate that Treg reconstitution post-HSCT is characterized by high levels of peripheral proliferation, which appear to be driven primarily by persistent CD4 T lymphopenia. However, post-HSCT Treg are also highly sensitive to FAS-mediated apoptosis. This process does not affect the survival of other CD4 T cell subsets. In the absence of thymic generation of Treg from hematopoietic precursors, this dynamic process results in a relative deficiency of Treg post-HSCT. Our findings provide important information for developing strategies aimed at monitoring and modulating Treg to promote immune tolerance following HSCT.

Prolonged Thrombocytopenia Is Associated With Increases Of CD8+ CX3CR1+ Cells In The Bone Marrow After Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5494-5494
Author(s):  
Xiaohui Zhang ◽  
Guoxiang Wang ◽  
Honghu Zhu ◽  
Yanrong Liu ◽  
Daihong Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Cd8 T Cells ◽  
Hematopoietic Stem ◽  
The Impact

Abstract Prolonged thrombocytopenia is a common complication after allogeneic hematopoietic stem cell transplantation(allo-HSCT),which is associated with a high mortality and poor prognosis. The aim of this study was to assess the impact of the CD8+CX3CR1+ T cells on the development and maturation of megakaryocytes in patients with the prolonged thrombocytopenia after allo-HSCT in order to identify the risk factors related to thrombocytopenia after allo-HSCT. The changes in CD8+ T cells and their homing receptors CX3CR1, CXCR4 and VLA-4 in bone marrow of patients( N=89) with and without (N=94 ) prolonged thrombocytopenia following allo-HSCT and the impact of activated CD8+ T cells on apoptosis and ploidy of megakaryocytes in vitro ware determined. The percentage of CD8+CX3CR1+ T cells was significantly higher in prolonged thrombocytopenia patients than control (P<0.001).The increase in CD8+ T cells was not observed in peripheral blood. Patients with prolonged thrombocytopenia exhibited a marked increase in the number of low ploidy(≤8) megakaryocytes compared to those without(P<0.05). Depletion of CD8+ T cells increased apoptosis of megakaryocytes (P<0.05), which was corrected by reconstitution of CD8+ T cells (P<0.05). We demonstrated that prolonged thrombocytopenia is associated with increased expression of CX3CR1 and recruitment of CD8+ T cells into the bone marrow. Activated CD8+ T cells suppress apoptosis of megakaryocytes in vitro. Our findings shed light on the prolonged thrombocytopenia is associated with increases of CD8+ CX3CR1+ cells in the bone marrow after allo-HSCT patients. Disclosures: No relevant conflicts of interest to declare.

Natural Killer Cell Reconstitution after Haploidentical Hematopoietic Stem Cell Transplantation with Post-Transplant Cyclophosphamide: Elimination of Donor-Derived Mature Alloreactive NK Cells, but Favorable Conditions for Adoptive Immunotherapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4567-4567
Author(s):  
Antonio Russo ◽  
Giacomo Oliveira ◽  
Sofia Berglund ◽  
Raffaella Greco ◽  
Valentina Gambacorta ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Nk Cells ◽  
Proliferating Cells ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Gvhd Prophylaxis

Abstract INTRODUCTION The use of high-dose cyclophosphamide as post-transplant Graft versus Host Disease (GvHD) prophylaxis has revolutionized haploidentical hematopoietic stem cell transplantation (haplo-HSCT), allowing the safe infusion of T cell replete grafts. The efficacy of post-transplant cyclophosphamide (PT-Cy) has its basis in its capacity to selectively eliminate proliferating cells, including alloreactive T cells. It is however to date unknown whether PT-Cy affects the reconstitution of Natural Killer (NK) cells, whose alloreactivity is known to play a major role in T cell-depleted haplo-HSCT. PATIENTS AND METHODS We analyzed the grafts and serial peripheral blood (PB) and bone marrow (BM) samples from 14 patients who received T cell replete haplo-HSCT followed by PT-Cy at the San Raffaele Scientific Institute, Milan (n=10, OSR) or the Johns Hopkins University, Baltimore (n=4, JHU). OSR patient received a myeloablative conditioning, PB stem cell grafts, and sirolimus and mycophenolate as pharmacological GvHD prophylaxis. JHU patients received the "classical" Baltimore nonmyeloablative conditioning, unmanipulated BM grafts, and tacrolimus and mycophenolate as pharmacological GvHD prophylaxis. To characterize NK cells reconstitution, we monitored absolute counts and employed a 27-marker flow cytometry panel with high dimensional single-cell analysis using the bh-SNE algorithm. We used intracellular staining to determine the frequency of Ki67+ proliferating cells and expression of Aldehyde Dehydrogenase (ALDH), known to confer resistance to PT-Cy. Interleukin-15 (IL-15) serum concentration was quantified using the Bio-Plex Pro Human Cytokine 4-plex assay. To directly assess the effect of PT-Cy on proliferating NK cells we exposed graft NK cells to IL-15 and mafosfamide, a cyclophosphamide analogue active in vitro. Functional assays against leukemic cell lines and primary AML blasts were performed measuring CD107A degranulation on NK cells and Annexin V on targets. RESULTS All patients received high numbers of mature donor NK cells as part of the graft (OSR median 17x106/kg, JHU median 7.25x106/kg ), and donor-derived NK cells were detectable as early as day 3 after HSCT and throughout the entire follow-up. At day 3 after HSCT, all subsets of NK cells, including single KIR+ alloreactive cells, were actively proliferating (mean 61.23% of Ki-67+ cells for OSR patients, and 58% for JHU patients), possibly driven by the high levels of IL-15 detected in patient serum after conditioning (Fig.1A). After PT-Cy, a marked reduction in the frequency and counts of proliferating NK cells was evident irrespectively of the transplantation platform, suggesting selective killing of dividing cells by PT-Cy. In line with this hypothesis, NK cells from the graft and from patient PB at day 3 after HSCT showed no detectable ALDH expression, and NK cells prompted to proliferate in vitro were killed in a dose-dependent manner by mafosfamide (Fig.1B). The phenotype of NK cells also changed upon PT-Cy: whereas before the infusion they resembled their mature counterparts from the graft, after PT-Cy an immature phenotype, CD62L+NKG2A+KIR-, became prevalent, suggesting derivation from donor HSCs rather than from infused NK cells (Fig.1C). Accordingly, bhSNE maps demonstrated differential clustering of NK cells from the graft and analyzed 30 days after HSCT (Fig.1D). In line with these features, we detected very low numbers of putatively alloreactive single KIR+ NK cells both in the PB and in the BM of patients at day 30 after HSCT, and these NK cells displayed impaired cytotoxic potential against leukemic targets. Finally, consistent with these observations, when we analyzed the impact of predicted NK alloreactivity in an extended series of 99 patients who received myeloablative haplo-HSCT with PT-Cy, we detected no significant difference in progression-free survival (Fig.1E). CONCLUSION Our data suggest that the majority of mature NK cells infused with unmanipulated grafts are eliminated upon PT-Cy administration and that in this transplantation platform NK cell alloreactivity might be blunted by the elimination of donor single KIR+ NK cells and by the competition between reconstituting NK and T cells. Still, the high levels of IL-15 detected in patients' sera at early time-points might provide a biological rationale for the infusion of mature donor NK cells early after PT-Cy administration. Figure 1 Figure 1. Disclosures Bonini: TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy. Ciceri:MolMed SpA: Consultancy.

scholarly journals HLA-DPB1 Reactive T Cell Receptors for Adoptive Immunotherapy in Allogeneic Stem Cell Transplantation

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1264 ◽  
Author(s):  
Sebastian Klobuch ◽  
Kathrin Hammon ◽  
Sarah Vatter-Leising ◽  
Elisabeth Neidlinger ◽  
Michael Zwerger ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cd8 T Cells ◽  
T Cell Receptors ◽  
Hematopoietic Cells ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Receptors

HLA-DPB1 antigens are mismatched in about 80% of allogeneic hematopoietic stem cell transplantations from HLA 10/10 matched unrelated donors and were shown to be associated with a decreased risk of leukemia relapse. We recently developed a reliable in vitro method to generate HLA-DPB1 mismatch-reactive CD4 T-cell clones from allogeneic donors. Here, we isolated HLA-DPB1 specific T cell receptors (TCR DP) and used them either as wild-type or genetically optimized receptors to analyze in detail the reactivity of transduced CD4 and CD8 T cells toward primary AML blasts. While both CD4 and CD8 T cells showed strong AML reactivity in vitro, only CD4 T cells were able to effectively eliminate leukemia blasts in AML engrafted NOD/SCID/IL2Rγc−/− (NSG) mice. Further analysis showed that optimized TCR DP and under some conditions wild-type TCR DP also mediated reactivity to non-hematopoietic cells like fibroblasts or tumor cell lines after HLA-DP upregulation. In conclusion, T cells engineered with selected allo-HLA-DPB1 specific TCRs might be powerful off-the-shelf reagents in allogeneic T-cell therapy of leukemia. However, because of frequent (common) cross-reactivity to non-hematopoietic cells with optimized TCR DP T cells, safety mechanisms are mandatory.

scholarly journals BCG vaccination induces enhanced frequencies of memory T cells and altered plasma levels of common γc cytokines in elderly individuals

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0258743
Author(s):  
Nathella Pavan Kumar ◽  
Chandrasekaran Padmapriyadarsini ◽  
Anuradha Rajamanickam ◽  
Perumal Kannabiran Bhavani ◽  
Arul Nancy ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cd8 T Cells ◽  
Plasma Levels ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Elderly Individuals ◽  
Bcg Vaccination ◽  
Adaptive Immune

BCG vaccination is known to induce innate immune memory, which confers protection against heterologous infections. However, the effect of BCG vaccination on the conventional adaptive immune cells subsets is not well characterized. We investigated the impact of BCG vaccination on the frequencies of T cell subsets and common gamma c (γc) cytokines in a group of healthy elderly individuals (age 60–80 years) at one month post vaccination as part of our clinical study to examine the effect of BCG on COVID-19. Our results demonstrate that BCG vaccination induced enhanced frequencies of central (p<0.0001) and effector memory (p<0.0001) CD4+ T cells and diminished frequencies of naïve (p<0.0001), transitional memory (p<0.0001), stem cell memory (p = 0.0001) CD4+ T cells and regulatory T cells. In addition, BCG vaccination induced enhanced frequencies of central (p = 0.0008), effector (p<0.0001) and terminal effector memory (p<0.0001) CD8+ T cells and diminished frequencies of naïve (p<0.0001), transitional memory (p<0.0001) and stem cell memory (p = 0.0034) CD8+T cells. BCG vaccination also induced enhanced plasma levels of IL-7 (p<0.0001) and IL-15 (p = 0.0020) but diminished levels of IL-2 (p = 0.0033) and IL-21 (p = 0.0020). Thus, BCG vaccination was associated with enhanced memory T cell subsets as well as memory enhancing γc cytokines in elderly individuals, suggesting its ability to induce non-specific adaptive immune responses.

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