Increased Circulating of CD54highCD181low Neutrophils in Myelodysplastic Syndrome

Myelodysplastic syndromes (MDSs) are a group of heterogeneous hematopoietic stem/progenitor cells clonal diseases, characteristic features with myeloid dysplasia, leading to abnormality of neutrophils. Recent studied have showed that neutrophils act not only as professional killers, but also as regulators of innate and adaptive immune in infection and inflammatory condition. The CD54highCD181low neutrophils are a kind of reverse-transmigrated neutrophils characterized proinflammatory phenotype. We investigated the frequency and functional properties of circulating CD54highCD181low neutrophils in patients with untreated MDS. Frequency of CD54highCD181low neutrophils was significantly increased in MDS patients and related to the severity of the disease. Furthermore, CD54highCD181low neutrophils suppressed CD8+ T cells functions in vitro. CD54highCD181low neutrophils lead to upregulation of PD1 on CD8+ T cells. Higher CD54highCD181low neutrophils were related to poor prognosis and more infections. The frequency of CD54highCD181low neutrophils decreased in high risk MDS patients who had response after treatment with decitabine. Overall, we identified CD54highCD181low neutrophils expanded in MDS. The exact mechanisms of increased CD54highCD181low neutrophils and its effect on immune function remain to be elucidated.

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Transcription factor Etv6 regulates functional differentiation of cross-presenting classical dendritic cells

Journal of Experimental Medicine ◽

10.1084/jem.20172323 ◽

2018 ◽

Vol 215 (9) ◽

pp. 2265-2278 ◽

Cited By ~ 10

Author(s):

Colleen M. Lau ◽

Ioanna Tiniakou ◽

Oriana A. Perez ◽

Margaret E. Kirkling ◽

George S. Yap ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Transcription Factor ◽

T Cell ◽

Cd8 T Cells ◽

Functional Differentiation ◽

Specific Gene ◽

Hematopoietic Stem

An IRF8-dependent subset of conventional dendritic cells (cDCs), termed cDC1, effectively cross-primes CD8+ T cells and facilitates tumor-specific T cell responses. Etv6 is an ETS family transcription factor that controls hematopoietic stem and progenitor cell (HSPC) function and thrombopoiesis. We report that like HSPCs, cDCs express Etv6, but not its antagonist, ETS1, whereas interferon-producing plasmacytoid dendritic cells (pDCs) express both factors. Deletion of Etv6 in the bone marrow impaired the generation of cDC1-like cells in vitro and abolished the expression of signature marker CD8α on cDC1 in vivo. Moreover, Etv6-deficient primary cDC1 showed a partial reduction of cDC-specific and cDC1-specific gene expression and chromatin signatures and an aberrant up-regulation of pDC-specific signatures. Accordingly, DC-specific Etv6 deletion impaired CD8+ T cell cross-priming and the generation of tumor antigen–specific CD8+ T cells. Thus, Etv6 optimizes the resolution of cDC1 and pDC expression programs and the functional fitness of cDC1, thereby facilitating T cell cross-priming and tumor-specific responses.

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

Blood ◽

10.1182/blood.v120.21.3077.3077 ◽

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.

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Dysregulated T Cell Response in Bone Marrow Immune Micro-Environment of Patients with Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v126.23.3140.3140 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3140-3140

Author(s):

Yu-tong Wang ◽

Yuan Kong ◽

Yang Song ◽

Zheng-Fan Jiang ◽

Xiao-jun Huang

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cell Response ◽

Graft Function ◽

T Cell Response ◽

Intracellular Cytokine ◽

Hematopoietic Stem ◽

Poor Graft Function

Abstract Background: Poor graft function (PGF), a kind of bone marrow (BM) failure syndrome, is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nevertheless, the exact mechanisms underlying PGF remain unclear. The BM immune micro-environment is considered to be involved in the regulation of murine hematopoiesis. Dysregulated T cell response was found to suppress proliferation and induce apoptosis of hematopoietic progenitor cells in patients with aplastic anemia. Therefore, we conducted a study to analyze the alteration of T cell subpopulations in BM micro-environment of allotransplant patients. Aims: To compare the cellular compositions and function of T cells in BM micro-environment between patients with PGF and good graft function (GGF) after allo-HSCT in Peking University Institute of Hematology. Methods: Using a prospective nested case-control study, the active phenotype and memory phenotype of CD4+ T cells and CD8+ T cells in BM were analyzed by flow cytometry in 12 patients with PGF, 36 matched patients with GGF after allo-HSCT, and 15 healthy donors (HDs). Furthermore, the cytokine secretion function of CD4+ T cells and CD8+ T cells were evaluated after simulation and the level of eight Th1 and Th2 cytokines in BM plasma were detection by cytometric beads assay. Results: The demographic and clinical characteristics were similar between allo-HSCT patients with PGF and those with GGF. Although the PGF patients presented a significant lymphopenia, a notable increased percentage of activated CD8+ T cells was detected in the BM of PGF patients when compared to that in GGF patients (61.7% versus 35.0%, P =.02). Moreover, the in vitro cytokine stimulated tests demonstrated a significant higher proportion of Tc1 in PGF patients (46.1% versus 20.3% versus 28.4%, P <.005), an elevated percentage of Th1 in PGF compared with HDs (38.5% versus 21.7%, P <.005), a higher percentage of Th2 (4.5% versus 2.1% versus 2.3%, P <.005) and a dramatically decreased percentage of Tc2 in PGF (0.6% versus 2.0% versus 2.0%, P <.0001). Therefore, a significant elevation in the ratio of Th1/Th2 (19.73 versus 7.39 versus 6.91, P <.0001) and Tc1/Tc2 (67.25 versus 10.07 versus 14.57, P <.005) were observed in PGF when compared with those in GGF and HDs. The changes of IFN-gama and IL-4 levels in BM plasma detected by cytometric beads assay were in accordance with the intracellular cytokine results analyzed by flow cytometry. Summary/Conclusion: Both the in vitro intracellular cytokine testing after stimulation and the BM plasma cytokine detection provides evidence that CD4+ and CD8+ T cells were polarized towards a type-1 cytokine response in patients with PGF, suggesting that the dysfunction of T cell response in BM immune micro-environment may hamper the hematopoietic recovery after allo-HSCT. Acknowledgment: Supported by the National Natural Science Foundation of China (grant nos. 81370638&81230013), and the Beijing Municipal Science and Technology Program (grant nos. Z141100000214011& Z151100004015164& Z151100001615020). Disclosures No relevant conflicts of interest to declare.

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Adenovirus Specific T-Cell Lines Devoid of Alloreactivity Against Haploidentical Recipients Can Be Obtained Using a Set of Adenovirus Hexon Peptides.

Blood ◽

10.1182/blood.v110.11.3273.3273 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3273-3273

Author(s):

Patrizia Comoli ◽

Marco W. Schilham ◽

Sabrina Basso ◽

Tamara van Vreeswijk ◽

Rita Maccario ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Cd8 T Cells ◽

Peptide Pool ◽

Specific Lysis ◽

Hematopoietic Stem ◽

Hexon Protein ◽

T Cell Lines

Abstract Human Adenovirus (HAdV) infection/reactivation may cause life-threatening complications in recipients of hematopoietic stem cell transplantation (HSCT), the highest risk being observed in pediatric recipients of a T-cell depleted allograft from haploidentical family donor. The effectiveness of pharmacological therapy for HAdV infection is still suboptimal. It has been recently demonstrated that cell therapy may offer a unique opportunity to restore antiviral immune surveillance, leading to clearance of infection and prevention/treatment of disease. However, infusion of HAdV-specific T-cells in the haplo-HSCT cohort poses the concern that GVHD may ensue as a consequence of T-cell transfer. We have conducted scale-up experiments to validate a method of in vitro culture to expand T-cells specific for HAdV, based on stimulation of donor peripheral blood mononuclear cells (PBMC) with a pool of 5 30-mer peptides derived from HAdV5 hexon protein, for use in recipients of haplo-HSCT (Veltrop-Duits et al, Eur J Immunol36, p2410; 2006). A total of 20 T-cell lines were generated, starting from a median of 20 × 106 donor PBMC, that yielded a median of 80 × 106 cells. Most of the cell lines obtained included a majority of CD4+ T-lymphocytes, with a lower % CD8+ cells (median and range: 78, 19–94 and 18, 5–58, respectively) but 5/20 lines contained a high number of CD8+ T cells (ranging between 43% and 58%), which were CD56+ and/or TCRγδ+, and in 1 case also 44% NK cells. Eighteen of the 20 T-cell lines were HAdV-specific, since they showed a median proliferation to the HAdV hexon peptide pool and inactivated HAdV of 14615 (95%CI 8924–31532) and 11103 (95%CI 8805–30174) cpm/105 cells after subtraction of background (responders+irradiated autologous PBMC), respectively. HAdV-specific lysis >10% at a 2:1 effector to target (E:T) ratio was observed in 50% of the T-cell lines. The 2 non-specific, as well as the 3 T-cell lines with lower specific activity, included >40% CD8+ T-cells. Production of IFNγ in an ELIspot assay to HAdV hexon peptide pool above 40 SFU/105 cells was observed in 9 out of 13 tested T-cell lines. Evaluation of specific response to hexon peptides in showed a majority of responses to II42 (80%), with 50–60% responses to II50, II57, II61, and II64. Only 2 out of the 20 T-cell lines tested were prevalently alloreactive against the recipient. Of the 18 HAdV-specific lines, 1 showed higher proliferation to patient PBMC than to HAdV (13518 vs 11717 mean cpm), and would have thus been discarded as unsuitable for in vivo use, while the other 17 showed no alloreactivity (14) or alloreactivity between 10 and 23% of specific proliferation (3). None of these 18 T-cell lines showed lysis >5% against recipient PHA blasts in the cytotoxicity assay. Our data show that PBMC stimulation with HAdV hexon protein-derived 30-mer peptides is able to reproducibly induce the generation of HAdV-specific CD4+ T-cell lines with efficient in vitro antiviral response in most HLA-mismatched HSCT donors. The majority of these T-cell lines show low/undetectable alloreactivity against recipient targets, and could therefore be safely employed for adoptive treatment of HAdV complications developing after HSCT from a HLA-haploidentical donor.

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Development of T-Cell Therapy Targeting Hematopoietic Minor Histocompatibility Antigen HA-1

Blood ◽

10.1182/blood-2019-130552 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5749-5749

Author(s):

Artem Pilunov ◽

Dmitrii Romaniuk ◽

Savely Sheetikov ◽

Alexandra Khmelevskaya ◽

Anton Shmelev ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Conflicts Of Interest ◽

Significant Proportion ◽

Published Data ◽

Functional Screening ◽

Hematopoietic Stem ◽

Normalized Frequency

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently the only curative therapy for hematological malignancies yet in nearly one-third of patients, it is followed by a relapse of the disease contributing to high mortality. In fully HLA-matched allo-HSCT graft versus leukemia reaction is driven by the recognition of the minor histocompatibility antigens (MiHAs) - endogenous polymorphic peptides presented by MHC. Particularly, HA-1 MiHA is a promising target for immunotherapy. HA-1 is presented by frequent among Caucasians HLA allele - A*02:01. The single nucleotide variation in ARGHAP45 gene which generates the MiHA has the optimal allelic distribution, thus immunogenic mismatch occurs in 30% of allo-HSCT. Also, ARGHAP45 is overexpressed in certain types of leukemia. Here we aim to develop HA-1-specific T-cells for post-transplant relapse therapy. To obtain the sequences of HA-1-specific T-cell receptors (TCRs), naive CD8+ T-cells from 3 HLA-A*02:01 positive and HA-1 negative donors were expanded in vitro using autologous dendritic cells pulsed with HA-1 peptide. Antigen-specific cells were enriched by CD137 marker expression or by HLA-tetramer staining, RNA from positive and negative fractions was isolated for cDNA library preparation. The α and β TCR-repertoires were sequenced using the Illumina MiSeq system. The representative enrichment plot is shown in Figure 1 (A - α chains, B - β chains). Each circle represents a unique TCR. The vertical axis shows the normalized frequency in enriched fraction, the horizontal axis shows the normalized frequency in tetramer or CD137 negative flowthrough. HA-1-specific TCRs are denoted by green filled circles.TCRs were considered to be HA-1-specific if they were significantly enriched in CD137+ or tetramer+ fraction (exact Fisher test, p=0.05). In total, 49 α and 80 β chains were described. To determine the degree of similarity between HA-1-specific TCRs Levenstein distance was calculated between amino acid sequences of complementarity-determining region 3 for both chains. Sequences of previously published HA-1-specific TCRs were also included in the analysis (Verdijk et.al., Haematologica, 2002; Bleakley et.al., 2017, WO2018058002A1). α chains demonstrated low degree of mutual similarity, the majority of sequences did not belong to any cluster (Figure 2A, sequences with the Levenstein distance <3 are connected). In contrast, a significant proportion of β chains were organized in a few clusters containing sequences from all 3 donors and previously published data (Figure 2B). We selected 14 α and 12 β HA-1-specific TCR chains (marked by the black dots in Figure 2). Clones were picked to represent separate clusters of similarity to Levenstein metrics, and unique sequences. Selected α and β-chains were cloned for subsequent functional screening in different combinations. Besides, we developed the modular lentiviral backbone for manufacturing HA-1 specific transgenic CD8+ T-cells. Our approach utilizes Golden Gate Cloning, which allows rapid assembly of lentiviral backbone carrying any combination of TCR α and β chains fused with the selective marker for sorting via p2A peptides. We used truncated CD34 as a transduced cell surface marker for the rapid separation of transduced cells by clinical-grade antibodies and subsequent expansion. In order to prevent the mispairing of transgenic TCR with endogenous one, CRISPR/Cas9 knockout strategy of endogenous TCR chains was developed. We used guide RNAs specific to TRAC,TRBC1 and TRBC2 genes and recombinant Cas9. The efficiency was demonstrated on Jurkat E6-1 cell line, the knockout was confirmed both by flow cytometry and genotyping of the modified cells using fragment analysis. Constant regions of the transgenic TCRs were modified to prevent cleavage by Cas9, the resistance was confirmed by in vitro Cas9 digestion assay. Moreover additional cysteines were introduced in the constant regions of transgenic TCRs for increased transgenic TCR stability. Cytotoxic activity of modified cells will be confirmed on lymphoblastoid cell lines and patient leukemia samples, cytokine secretion of modified cells will be detected using ELISPOT. The work was supported by the Russian Foundation for Basic Research grant 19-29-04156. Disclosures No relevant conflicts of interest to declare.

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Human CD8 T cells generated in vitro from hematopoietic stem cells are functionally mature

BMC Immunology ◽

10.1186/1471-2172-12-22 ◽

2011 ◽

Vol 12 (1) ◽

pp. 22 ◽

Cited By ~ 26

Author(s):

Génève Awong ◽

Elaine Herer ◽

Ross N La Motte-Mohs ◽

Juan Zúñiga-Pflücker

Keyword(s):

Stem Cells ◽

T Cells ◽

Hematopoietic Stem Cells ◽

Cd8 T Cells ◽

Hematopoietic Stem

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

Blood ◽

10.1182/blood.v114.22.2438.2438 ◽

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.

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

Blood ◽

10.1182/blood.v122.21.5494.5494 ◽

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.

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Tumor-Specific Donor Lymphocyte Infusion for Tumor Relapse after MHC-Haploidentical Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v128.22.2157.2157 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2157-2157

Author(s):

Isao Tawara ◽

Kana Okamori ◽

Naoyuki Katayama ◽

Hiroshi Shiku ◽

Hiroaki Ikeda

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Growth ◽

Cd8 T Cells ◽

Research Funding ◽

Cd8 T Cell ◽

Donor Lymphocyte Infusion ◽

Hematopoietic Stem ◽

Tumor Relapse

Abstract Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for refractory hematologic malignancies such as leukemia and myelodysplastic syndrome. Tumor relapse, graft-vs host disease (GVHD) and infections are, however, major obstacles to successful allo-HSCT. Donor lymphocyte infusion (DLI) is carried out in some cases of tumor relapse and infections after allo-HSCT. DLI potentially induces or aggravates GVHD due to allo-reactivity of the lymphocytes used for infusion and the efficacy of DLI for tumor relapse is actually limited. Therefore, development of the method of DLI with enhanced anti-tumor effects without induction or aggravation of GVHD is expected to improve the results of DLI in the context of tumor relapse. Tumor antigen-specific T cell receptor (TCR)-expressing cell infusion is one of the potentially effective immunotherapies for refractory tumors. We previously reported that tumor antigen-specific TCR-gene-transduced human lymphocytes engineered with a novel retrovirus vector silencing endogenous TCRs showed reduced allo-reactivity (ASH annual meeting 2014). Utilizing this technology, we may be able to enhance anti-tumor effects without induction or aggravation of GVHD. In this study, we conducted a mouse pre-clinical model that mimics tumor-specific TCR-engineered DLI for tumor relapse after MHC-haploidentical HSCT and explored the effect of DLI on tumor growth and GVHD. We performed experimental study utilizing the following model: MHC-haploidentical BALB/c(H-2d)→CB6F1(H-2b/d) HSCT, BALB/c-derived sarcoma CMS5a and T cells from TCR-transgenic mouse DUC18. CMS5a has a unique mutated form of a mitogen-activated kinase ERK2, and a nonamer peptide, called 9m, incorporating the resulting amino acid substitution is presented on H-2Kd and recognized by the CD8+ CTL clone C18. DUC18 is a BALB/c-background TCR transgenic line that expresses the rearranged Vα10.1 and Vβ8.3 genes of the C18. CD8+ T cells of DUC18 mice can suppress CMS5a tumor growth in the syngeneic BALB/c hosts. Allo-reactivity of DUC18 T cells was reduced as compared to that of wild type BALB/c T cells. Based on the results of preliminary experiments, we set standard experimental conditions as follows: X-ray irradiation (10 Gy) for pre-conditioning, 5x106 whole bone marrow and 0.25x106 T cell transplant (BMT) for mild GVHD induction, 1x106 CMS5a inoculation for progressive tumor growth in BMT recipients, and 4x106 CD8+ T cells for observation of the effect of DLI on tumor growth and GVHD. In some experiment, CD4+ T cells were co-infused with CD8+ T cells. DLI was carried out 3 days after CMS5a inoculation at 2, 4 or 8 weeks after BMT. We first used CD8+ T cells from naïve BALB/c or DUC18 mice for DLI at 8 weeks after BMT. No GVHD aggravation was observed in either BALB/c or DUC18 CD8+ T cell recipients and, as expected, suppression of tumor growth was observed only in the DUC18 CD8+ T cell recipients. Next, DLIs using CD8+ T cells only, or both CD8+ and CD4+ (CD8+/CD4+) T cells from naïve mice were carried out at 4 or 2 weeks after BMT. No apparent effect of CD4+ T cell co-infusion on tumor growth and GVHD was observed in both types of donor T cell recipients when DLI was carried out at 4 weeks after BMT. However, GVHD was aggravated in CD8+/CD4+ T cell recipients from both types of donors when DLI was carried out at 2 weeks after BMT, and no CD4-driven additional anti-tumor effect was observed in DUC18 T cell recipients. We then performed DLI using in vitro activated/expanded T cells including both CD8+ and CD4+ as a more clinically relevant model. BALB/c and DUC18 splenocytes were activated and expanded in vitro and infused (adjusted CD8+ T cell number to 4x106/mouse) into recipients at 2 or 8 weeks after BMT. Aggravation of GVHD was observed in either BALB/c or DUC18 activated/expanded T cell recipients when DLI was carried out at 2 weeks after BMT. On the other hand, it was not observed when DLI was carried out at 8 weeks after BMT. Anti-tumor effect of DUC18 T cells was observed regardless of timings of DLI. Taken together, infusion of tumor-specific donor lymphocytes with reduced allo-reactivity for tumor relapse after MHC-haploidentical HSCT will be promising strategy. Timing of DLI and condition of recipients such as inflammatory status may affect GVHD. Multiple model studies are required for development of more effective DLI strategies without aggravation of GVHD. Disclosures Tawara: Astellas: Honoraria. Katayama:Nippon Shinyaku: Honoraria; Chugai: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria; Dainippon Sumitomo Pharma: Honoraria; Pfizer: Honoraria; Celgene: Honoraria; Shionogi: Honoraria; Kyowa Hakko Kirin: Honoraria, Research Funding; Taisho Toyama Pharma: Honoraria; Shire: Honoraria; Astellas: Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Eisai: Honoraria; Takeda: Honoraria; Bristol-Myers Squibb Japan: Honoraria. Ikeda:Ono Pharmaceutical Co., Ltd: Honoraria; Daiichi Sankyo Co., Ltd: Honoraria.

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

Cells ◽

10.3390/cells9051264 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1264 ◽

Cited By ~ 3

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.

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