scholarly journals Alterations of Peripheral Blood T Cell Subsets following Donor Lymphocyte Infusion in Patients after Allogeneic Stem Cell Transplantation

Hemato ◽  
2021 ◽  
Vol 2 (4) ◽  
pp. 692-702
Author(s):  
Ann-Kristin Schmaelter ◽  
Johanna Waidhauser ◽  
Dina Kaiser ◽  
Tatjana Lenskaja ◽  
Stefanie Gruetzner ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Peripheral Blood ◽  
Donor Lymphocyte Infusion ◽  
T Cell Subsets ◽  
Mixed Chimerism

Donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation (alloSCT) is an established method to enhance the Graft-versus-Leukemia (GvL) effect. However, alterations of cellular subsets in the peripheral blood of DLI recipients have not been studied. We investigated the changes in lymphocyte subpopulations in 16 patients receiving DLI after successful alloSCT. Up to three DLIs were applied in escalating doses, prophylactically for relapse prevention in high-risk disease (n = 5), preemptively for mixed chimerism and/or a molecular relapse/persistence (n = 8), or as part of treatment for hematological relapse (n = 3). We used immunophenotyping to measure the absolute numbers of CD4+, CD8+, NK, and CD56+ T cells and their respective subsets in patients’ peripheral blood one day before DLI (d-1) and compared the results at day + 1 and + 7 post DLI to the values before DLI. After the administration of 1 × 106 CD3+ cells/kg body weight, we observed an overall increase in the CD8+ and CD56+ T cell counts. We determined significant changes between day − 1 compared to day + 1 and day + 7 in memory and activated CD8+ subsets and CD56+ T cells. Applying a higher dose of DLI (5 × 106 CD3+ cells/kg) led to a significant increase in the overall counts and subsets of CD8+, CD4+, and NK cells. In conclusion, serial immune phenotyping in the peripheral blood of DLI recipients revealed significant changes in immune effector cells, in particular for various CD8+ T cell subtypes, indicating proliferation and differentiation.

scholarly journals Neoantigen Landscape of Relapsed Acute Leukemia Following Allogeneic Stem Cell Transplantation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4595-4595
Author(s):  
Karolyn Oetjen ◽  
Cai Chen ◽  
Christian Bradley ◽  
Reema Panjwani ◽  
Cheng Yan ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Acute Leukemia ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Peripheral Blood ◽  
Somatic Mutations ◽  
Class I

Abstract INTRODUCTION: The potent graft versus leukemia (GVL) effect of allogeneic stem cell transplantation (allo-SCT) is considered as a blueprint for cellular immunotherapy. However, failure of GVL leads to relapse of underlying leukemia, the major cause of death after allo-SCT. In solid tumors, higher tumor mutation burdens are associated with better response to check point inhibitors which implies the importance of neoantigen specific T-cell functions in cancer immunity. In contrast, the frequencies of somatic mutations in acute leukemia are generally low, therefore the role of neoantigens in GVL remains undetermined. Here, we developed a platform to screen for potential neoantigens by performing whole exome sequencing (WES) and RNA sequencing (RNAseq) in matched samples: leukemic blasts at relapse after allo-SCT, recipient T cell controls, and donor cells. METHODS: Leukemic blasts from patients in relapse were enriched by flow sorting from bone marrow aspirate or peripheral blood samples. Recipient T cells were isolated from pre-transplant peripheral blood as germline controls, and donor monocytes or CD34-positive cells were used as hematopoietic-lineage cell controls. WES was performed to 100X coverage, paired with RNAseq 40M reads per sample. Somatic mutations were detected with mutect and mpileup, followed by annotation with SnpEff. High confidence somatic mutations were subjected to pVAC-seq for neoantigen predictions. RESULTS: Six patients with relapsed acute leukemia (AML 5, ALL 1) after allo-SCT and their transplant donors (matched sibling 3, haplo-identical 3) had suitable samples available for analysis. On average, somatic mutations were identified in 297 genes (range 108- 609) by comparing leukemic blasts and germline control T cells. Among those mutations, potential candidates of neoantigen were identified in five out of six subjects. Allele frequencies of mutant genes varied. Most of neoantigens were predicted to bind HLA of both class I (median 5, range 0-15) and class II (median 6, range 0-12). One subject had only HLA class II restricted peptides as predicted neoantigens. Of interest majority of antigens were derived from molecules known to play important roles in leukemia or tumor biology which include ETV6, CCNY, IDH2, PTPN11, SF3B1, and TP53. Evolution analysis of neoantigen showed an emergence of new antigens in relapsed leukemia while a few driver gene mutations persisted after allo-SCT (Figure). CONCLUSION: Our in-silico analysis demonstrated the possibility that somatic mutation in acute leukemia could serve as putative neoantigens applicable for novel immunotherapy after allo-SCT. The binding capacity of mutant peptides to class I and II HLA implies the importance of both CD4 and CD8 contributions to anti-neoantigen immunity. Next, we will search for neoantigen specific T cells exerting an anti-leukemia effect to validate the GVL potential of these mutations in allo-SCT. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Increasing Mixed Chimerism Is an Important Prognostic Factor for Unfavorable Outcome in Children With Acute Lymphoblastic Leukemia After Allogeneic Stem-Cell Transplantation: Possible Role For Pre-Emptive Immunotherapy?

2004 ◽  
Vol 22 (9) ◽  
pp. 1696-1705 ◽  
Author(s):  
Peter Bader ◽  
Hermann Kreyenberg ◽  
Walter Hoelle ◽  
Gregor Dueckers ◽  
Rupert Handgretinger ◽  
...  
Keyword(s):  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Lymphoblastic Leukemia ◽  
Donor Lymphocyte Infusion ◽  
Mixed Chimerism ◽  
Acute Leukemias ◽  
Low Level

Purpose We recently reported that children with acute leukemias who show increasing mixed chimerism (MC) after allogeneic stem-cell transplantation have a significantly enhanced risk of relapse. Here we present the results of a prospective multicenter study to investigate (1) whether relapse of acute lymphoblastic leukemia (ALL) can be determined in advance by serial analysis of chimerism, and (2) if outcome can be influenced by withdrawal of immunosuppression and/or by low-dose donor lymphocyte infusion when increasing MC is detected. Patients and Methods Serial and quantitative analysis of chimerism was performed using a fluorescent-based short-tandem-repeat–polymerase chain reaction in 163 children with ALL. Results One hundred one patients revealed complete chimerism (CC) or low-level MC (CC/low-level MC); increasing MC was found in 46 patients; and decreasing MC, in 16 patients. Relapse was significantly more frequent in patients with increasing MC (26 of 46) than in patients with CC/low-level MC (eight of 101) or in patients with decreasing MC (0 of 16; P < .0001). The probability of 3-year event-free survival (EFS) was 54% for all patients, 66% for patients with CC/low-level MC (n = 101), 66% for patients with decreasing MC (n = 16), and 23% for patients with increasing MC (n = 46; P < .0001). Of the 46 patients with increasing MC, 31 received immunotherapy. This group had a significantly higher 3-year EFS estimate (37%) than the 15 patients who did not receive immunotherapy (0%; P < .001). Conclusion Serial analysis of chimerism reliably identifies patients at highest risk to relapse. The 3-year EFS of patients with increasing MC without immunotherapy was 0%, by which overt relapse could be prevented in a considerable group of patients.

Generation and Functional Characterization of T Cells Against Candida albicans as Potential Immunotherapy after Allogeneic Stem Cell Transplantation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3226-3226 ◽  
Author(s):  
Thomas Lehrnbecher ◽  
Olaf Beck ◽  
Ulrike Koehl ◽  
Frauke Roeger ◽  
Klaus-Peter Hunfeld ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Candida Spp ◽  
Ifn Gamma ◽  
Cell Clones ◽  
Number Of Cells

Abstract Invasive fungal infections (IFI), in particular infections due to Aspergillus spp and Candida spp, still pose considerable problems in patients undergoing allogeneic stem cell transplantation (SCT). Despite the availability of new antifungal agents, morbidity and mortality of IFI are still unacceptable high. Although neutropenia is known as the single most important risk factor for IFI, there is a growing body of evidence that T cells play a major role in the defense against fungi. Therefore, adoptive immunotherapy with T cells against Candida spp. might be an interesting therapeutic option in patients undergoing allogeneic SCT. After overnight incubation of 1×108 peripheral blood mononuclear cells from 4 healthy individuals with cellular extracts of C.albicans, activated T cells were selected using the IFN-γ secretion-assay (Miltenyi Biotec, Bergisch Gladbach, Germany). After 14 days of culture, T cell clones were generated by limiting dilution and incubated for another 14 days. The median number of cells obtained was 2.6×107 (range, 0.85–5.75×107). Flow cytometry revealed a highly homogenous population of CD3+CD4+ cells (97.2% ± 2.6; n=6), of which an average of 8.6% (range, 4.8–58.2%) produced IFN-gamma on re-stimulation with C.albicans antigens, as assessed by intracellular cytokine staining assay. 20.5% (range, 5.8–72.4%) of the generated cells produced TNF-alpha, whereas no significant number of cells produced TH2 cytokines such as IL-4 and IL-10, indicating that the generated T cell clones were TH1 cells. The percentage of IFN-gamma producing T cells was significant upon stimulation with C.albicans and C.tropicalis, whereas less than 1% of cells produced IFN-gamma upon stimulation with antigens of other yeasts such as C.glabrata, Debaryomyces hansenii and Kluyveromyces lactis and molds such as A.fumigatus, Penicillium chrysogenum and Alternaria alternata. Compared to CD4+ T cells of the original fraction, the isolated and expanded anti-Candida T cells showed reduced alloreactivity, as assessed by means of CSFE. In addition, a strong proliferation of the generated anti-Candida T cells was seen after re-stimulation with C.albicans antigens. The potency of the generated T-cells to damage C.albicans was evaluated using the XTT assay. Compared to polymorphonucelar cells (PMNs), APCs and T-cells alone or to the combination of PMNs with T cells or APCs, respectively, the combination of PMNs, APCs and T-cells showed highest fungal damage (n=4). In conclusion, our data suggest that the isolation and expansion of anti-Candida T cells is possible and feasible. The generated T cells show low alloreactivity in vitro and increase the antimycotic potential of phagocytes. Thus, antimycotic T cells might become an important tool in the prophylaxis and therapy of IFI in patients after allogeneic SCT.

Recovery of KIR Expression After Allogeneic Stem Cell Transplantation in Multiple Myeloma Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4555-4555
Author(s):  
Thomas Stuebig ◽  
Michael Lioznov ◽  
Ulrike Fritsche-Friedland ◽  
Haefaa Alchalby ◽  
Christine Wolschke ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Nk Cells ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Time Points ◽  
Kir Receptors

Abstract Abstract 4555 Introduction: Activating and inhibitory killer immunoglobulin like receptors (KIR) are predominantly expressed on natural killer (NK) cells. KIR mismatch allogeneic stem cell transplantation (alloSCT) has been reported to provide beneficial effects for Multiple Myeloma (MM). However, their recovery in MM patients remains poorly understood. We, therefore, analysed KIR recovery in 90 MM patients after alloSCT. Methods: KIR expression (CD158a/h, CD158b/b2, CD158e1/e2) on NK cells and T cell subsets was measured by flow cytometry at different time points after alloSCT. Results: During the first 90 days after alloSCT NK cells represent the largest lymphocyte subset. Activating receptors like NKp30 and NKp44 showed a fluctuating expression while members of the KIR family were expressed at a constant rate (20% of NK cells). There was no significant difference in the early post transplantation period (day 0–90) compared to later time points (day 360). In contrast, T cells showed increased KIR expression during the first 30 days after alloSCT, which was highly significant for CD158e (p=0,0001). After 30 days the expression declined to baseline. Furthermore, T cell activation marker HLA-DR reached its highest expression between days 60 and 90 when KIR receptors were expressed at their lowest level (27% vs. 8%, p < 0,0001). Conclusions: We conclude that KIR receptors were differentially expressed on NK and T cells. Because KIR receptors are constantly expressed by NK cells and NK cells are the most frequent lymphocyte populations early after alloSCT, NK cells may be useful for KIR mismatch cellular therapy. Disclosures: No relevant conflicts of interest to declare.

Detection of Th1 Driven T-Cell Responses in Peripheral Could Guide Individualized Immunosuppression and Risk of Viral Reactivation Under GvHD Prophylaxis Following Allogeneic Stem Cell Transplantation.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3047-3047
Author(s):  
Judith Feucht ◽  
Kathrin Opherk ◽  
Cornelia Neinhaus ◽  
Simone Kayser ◽  
Wolfgang A. Bethge ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
T Cell Responses ◽  
Cell Responses ◽  
Cell Immunity

Abstract Abstract 3047 Allogeneic stem cell transplantation (SCT) can expose patients to a transient but marked immunosuppression, during which viral infections are an important cause of morbidity and mortality. The control of these infections will ultimately depend on the restoration of adequate T-cell immunity. Most viral infections after SCT are caused by endogenous reactivation of persistent pathogens such as cytomegalovirus (CMV), adenovirus (ADV) and Epstein-Barr-virus (EBV). Risk of viral complications is even higher under GvHD treatment or prophylaxis like calcineurin inhibitors and steroids. Post transplant often the immunosuppression needs to be reduced to improve viral complications with the risk of GvHD. The virus-specific T-cell responses in peripheral blood have been shown to be a good marker of immunological protection, but has not been used for clinical decision making and the guidance of drug plasma levels. Therefore, we performed a prospective clinical trial in 33 adult and pediatric patients after allogeneic stem cell transplantation receiving pharmacologic immunosuppression with steroids, Cyclosporin A, Tacrolimus, Everolimus or Mycophenolate. Median Age was 16 years. T-cell responses were analyzed ex vivo against Cytomegalovirus (pp65), Adenovirus (hexon antigen) and Epstein-Barr Virus (EBNA, LMP) using intracellular cytokine staining. In addition in vitro analysis of the proliferation responses using CFSE were performed. Responses were compared to healthy donors. The T-cell responses in vitro under low, high and supraphysiologic plasma concentrations of the respective drugs were investigated. Under the direct influence of steroids, activated, virus-specific T-cells underwent apoptosis. Among the Calcineurin inhibitors, Tacrolimus had the strongest inhibition on virus-specific T-cell immunity, followed by Cyclosporin A. But, under low therapeutic levels, Virus speciffic T-cell responses have been able to develop in PBMCs. Mycophenolate had only in high concentrations a strong effect on the T-cell response against viral pathogens. Relevant differences in the frequency of virus-specific T-cells secreting IFN-g could be detected within the CD4 compartment in correlation to the level of immunosuppression. In conclusion we could show that detection of virus-specific T-cells could be used to guide the level of immunosuppression in case of viral complications after allogeneic stem cell transplantation, since emergence of in vivo T-cell responses was closely associated with a clearance or reduction of the viral load. Disclosures: No relevant conflicts of interest to declare.

A Polyclonal Population of Piga Mutant CD52 and GPI Anchor Negative T Cells Can Give Early Immune Protection after Alemtuzumab-Based T Cell Depleted Allogeneic Stem Cell Transplantation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3134-3134
Author(s):  
Floris C. Loeff ◽  
J.H. Frederik Falkenburg ◽  
Lois Hageman ◽  
Sabrina A.J. Veld ◽  
Marian van de Meent ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Immune Protection ◽  
Coding Region ◽  
Membrane Expression ◽  
Gpi Anchor

Abstract Alemtuzumab, a monoclonal antibody targeting the glycophosphatidylinositol (GPI) anchored CD52 protein, is used for in vivo and/or in vitro T cell depletion before allogeneic stem cell transplantation (alloSCT) to reduce the risk of graft rejection and graft versus host disease. Following profound lymphodepletion, we observed a rapid recovery of T cell numbers early after transplantation despite presence of lytic levels of residual alemtuzumab (Halkes et al. ASH 2011). In the majority of patients, a substantial portion of these T cells completely lack CD52 membrane expression, explaining why these cells escaped alemtuzumab induced cytotoxicity. The aim of the current study was to further characterize these cells and unravel the mechanism underlying the loss of CD52 membrane expression. To study the functionality of the CD52 negative T cells, tetramer staining, cytokine production analysis, and cytotoxicity assays were performed. These analyses showed that the CD52 negative T cells which were present early after alloSCT contain functional T cells specific against multiple viral targets and that their lytic capacity was comparable to that of CD52 positive counterparts, demonstrating that the function of these cells is not impaired. To investigate whether absence of CD52 expression was the result of loss of CD52 gene expression, we performed mRNA expression analysis on CD52 negative T cells purified from peripheral blood samples taken from alloSCT recipients at three months post transplantation. No loss of CD52 mRNA expression was observed. Since CD52 is tethered to the membrane via the GPI anchor, we analyzed whether loss of CD52 membrane expression resulted from loss of GPI anchor expression by flow cytometry using counterstaining with a fluorescently labeled GPI-specific aearolysin FLAER. This analysis on CD52 negative T cells from 3 alloSCT recipients revealed that loss of CD52 expression generally resulted from loss of GPI anchor expression. To study whether loss of GPI anchor expression was due to active genetic down regulation, GPI positive and GPI negative CD8+ T cell populations (n=2) were purified by fluorescent activated cell sorting followed by gene expression analysis of the 26 genes that comprise the GPI anchor biosynthesis pathway. No overall loss of expression was observed for any of the 26 genes. Since loss of GPI anchor expression in paroxysmal nocturnal hemoglobinuria and aplastic anemia has been described to be the result of mutations in PIGA, one of the 26 GPI anchor biosynthesis genes and unique for its location on the X chromosome, we performed mutation analysis on clonally isolated and expanded CD52/GPI negative (CD4+ n=53, CD8+ n=13) and CD52/GPI positive (CD4+ n=8, CD8+ n=7) populations from 3 alloSCT patients. mRNA was isolated from each clone and Sanger sequencing was performed covering the protein coding region twice, with opposing primers. Mutations were only scored when observed in both reads. Using this strategy we were able to detect mutations in 35/53 CD4+ and in 8/13 CD8+ CD52/GPI negative clones, which included point mutations (n=7), non-coding mutations (n=5), small deletions <18bp (n=13), small insertions <5bp (n=6), and exon skipping (n=12). None of the individual mutations was found more than twice within clones from the same recipient, demonstrating a highly polyclonal mutational landscape. Additionally, in CD52/GPI positive clones no mutations in the PIGA coding region where found. To investigate whether these mutations in PIGA were sufficient to induce loss of GPI anchor expression, we analyzed 35 CD52/GPI negative CD4+ T cell clones by retroviral transduction with constructs encoding wtPIGA or an empty vector. Restored GPI anchor expression and coinciding CD52 membrane expression was observed in all 35 clones upon transduction with PIGA, but not with empty vector. We conclude that loss of CD52 membrane expression in T cells isolated early after alemtuzumab-based T cell depleted alloSCT is the result of various mutations in the PIGA gene and consequential loss of GPI anchor expression. We showed that these CD52 negative populations contain functional virus-specific T cells and may therefore be essential in immune protection early after transplantation. Disclosures Off Label Use: Alemtuzumab, conditioning of graft and/or recipient before allogeneic stem cell transplantation.

Sign in / Sign up

Export Citation Format

Share Document