scholarly journals The Role of γδ T Cells as a Line of Defense in Viral Infections after Allogeneic Stem Cell Transplantation: Opportunities and Challenges

Viruses ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 117
Author(s):  
Anke Janssen ◽  
Eline van Diest ◽  
Anna Vyborova ◽  
Lenneke Schrier ◽  
Anke Bruns ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Therapeutic Potential ◽  
Γδ T Cells ◽  
Full Potential ◽  
Γδ T Cell

In the complex interplay between inflammation and graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-HSCT), viral reactivations are often observed and cause substantial morbidity and mortality. As toxicity after allo-HSCT within the context of viral reactivations is mainly driven by αβ T cells, we describe that by delaying αβ T cell reconstitution through defined transplantation techniques, we can harvest the full potential of early reconstituting γδ T cells to control viral reactivations. We summarize evidence of how the γδ T cell repertoire is shaped by CMV and EBV reactivations after allo-HSCT, and their potential role in controlling the most important, but not all, viral reactivations. As most γδ T cells recognize their targets in an MHC-independent manner, γδ T cells not only have the potential to control viral reactivations but also to impact the underlying hematological malignancies. We also highlight the recently re-discovered ability to recognize classical HLA-molecules through a γδ T cell receptor, which also surprisingly do not associate with GVHD. Finally, we discuss the therapeutic potential of γδ T cells and their receptors within and outside the context of allo-HSCT, as well as the opportunities and challenges for developers and for payers.

Asymmetric Reconstitution of γδ T Cell Subsets after Haemopoietic Stem Cell Transplantation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2909-2909 ◽  
Author(s):  
Paul J. Travers ◽  
Andrea Knight ◽  
Sarah Grace ◽  
Panos Kottaridis ◽  
Stephen Mackinnon ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Γδ T Cells ◽  
Peripheral Circulation ◽  
Hemopoietic Stem Cell ◽  
Γδ T Cell ◽  
Post Transplant

Abstract In previous studies we and others have shown that the production of new T cells from the thymus declines after the third decade and that while patients below the age of 30 reconstitute primarily new T cells, those over thirty reconstitute primarily by expansion of pre-existing mature T cells. However little attention has been paid to the γδ subset of T cells, which form an important component of mucosal immune protection and which represent approximately 5% of peripheral T cells. Two major subsets of γδ T cells are defined by the expression of Vδ1 versus Vδ2, with Vδ1+ cells predominating in the fetal circulation and in mucosal sites, while Vδ2+ cells predominate in adult life and in the peripheral circulation. In light of the differerential preponderance of the two subsets in the fetal versus adult circulation, we have examined the reconstitution of these two subsets of γδ T cells following hemopoietic stem cell transplantation in a cohort of 28 patients sampled at 3 monthly intervals to ask whether both subsets recover adequately from adult stem cells. In 44 normal individuals, the median levels of Vδ1 and Vδ2 cells are 12.46 (0.22 to 167.8) and 32.78 (4.48 to 190.1) cells/mm3 respectively. In patients under 30, the reconstitution of the Vδ1 and Vδ2 subsets follow similar kinetics, reaching a plateau at 9 months post transplant with comparable numbers of Vδ1 and Vδ2 cells (note that the normal ratio of Vδ1 to Vδ2 is 0.38, so in the patients there is a significant increase in the proportion of Vδ1 cells in the peripheral circulation). In patients over 30 years of age, there is an even more significant disparity in the reconstitution of the two subsets. The Vδ1 subset recovers with similar kinetics as is seen in the patients under 30, although to slightly lower final levels. The Vδ2 subset, however, shows very little recover y, reaching a plateau at 6 months at the bottom of the normal range for up to 2 years post transplant. In these patients the ratio between Vδ1 and Vδ2 is inverted with an increasingly greater proportion of Vδ1 cells at longer times after transplant, with ratios in excess of 10 by 24 months post transplant, a 30-fold increase in the normal proportion of Vδ1 + T cells. Given that the Vδ1 subset shows a very restricted repertoire compared to the Vδ2 subset, the overall capacity of the circulating γδ T cell population to recognise and respond to antigen will be significantly compromised in older transplant recipients.

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.

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.

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.

The role of Vδ2-negative γδ T cells during cytomegalovirus reactivation in recipients of allogeneic stem cell transplantation

Blood ◽  
2010 ◽  
Vol 116 (12) ◽  
pp. 2164-2172 ◽  
Author(s):  
Andrea Knight ◽  
Alejandro J. Madrigal ◽  
Sarah Grace ◽  
Janani Sivakumaran ◽  
Panagiotis Kottaridis ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Γδ T Cells ◽  
Cell Receptor ◽  
Barr Virus ◽  
Cmv Reactivation

Abstract Reactivation of cytomegalovirus (CMV) remains a serious complication after allogeneic stem cell transplantation, but the role of γδ T cells is undefined. We have studied the immune reconstitution of Vδ2negative (Vδ2neg) γδ T cells, including Vδ1 and Vδ3 subsets and Vδ2positive (Vδ2pos) γδ T cells in 40 patients during the first 24 months after stem cell transplantation. Significant long-term expansions of Vδ2neg but not Vδ2pos γδ T cells were observed during CMV reactivation early after transplantation, suggesting direct involvement of γδ T cells in anti-CMV immune responses. Similarly, significantly higher numbers of Vδ2neg γδ T cells were detected in CMV-seropositive healthy persons compared with seronegative donors; the absolute numbers of Vδ2pos cells were not significantly different. The expansion of Vδ2neg γδ T cells appeared to be CMV-related because it was absent in CMV-negative/Epstein-Barr virus-positive patients. T-cell receptor-δ chain determining region 3 spectratyping of Vδ2neg γδ T cells in healthy subjects and patients showed restricted clonality. Polyclonal Vδ2neg cell lines generated from CMV-seropositive healthy donors and from a recipient of a graft from a CMV-positive donor lysed CMV-infected targets in all cases. Our study shows new evidence for role of γδ T cells in the immune response to CMV reactivation in transplantation recipients.

scholarly journals Reconstitution of T-cell-mediated immunity in patients after allogeneic stem cell transplantation

2020 ◽  
Vol 65 (1) ◽  
pp. 24-38
Author(s):  
N. N. Popova ◽  
V. G. Savchenko
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Cell Mediated Immunity ◽  
Term Survival ◽  
Post Transplant

Background. The timely reconstitution of the donor-derived immune system is a key factor in the prevention of such post-transplant complications as graft versus host disease, relapse or secondary tumours and various infections. These complications affect the long-term survival of patients after allogeneic stem cell transplantation.Aim — to describe the main stages of T Cell–mediated immune recovery in patients after allogeneic stem cell transplantation.General findings. T-cell–mediated immunity is responsible for anti-infective and anti-tumour immune response. The early post-transplant period is characterized by the thymus-independent pathway of T-cell recovery largely involving proliferation of mature donor T cells, which were transplanted to the patient together with hematopoietic stem cells. To a lesser extent, this recovery pathway is realized through the expansion of host naïve and memory T cells, which survived after conditioning. Thymus-dependent reconstitution involves generation of de novo naïve T cells and subsequent formation of a pool of memory T-cells providing the main immunological effects — graft versus tumour and graft versus host reactions. A better understanding of the T-cell immune reconstitution process is important for selecting optimized pre-transplant conditioning regimens and patient-specific immunosuppressive therapy approaches, thus reducing the risks of post-transplant complications and improving the long-term survival of patients after allogeneic stem cell transplantation.

Human Cytomegalovirus: Degranulation Is a Prominent Feature of Functionally Impaired pp65- and IE-1-Specific T-Cells in Patients after Allogeneic Stem Cell Transplantation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2572-2572
Author(s):  
Stephan Fuhrmann ◽  
Susanne Ganepola ◽  
Lutz Uharek ◽  
Eckhard Thiel ◽  
Wolf-Dieter Ludwig ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
High Risk ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Human Cytomegalovirus ◽  
Cd8 T Cells ◽  
Allogeneic Stem Cell Transplantation ◽  
Cmv Reactivation

Abstract Human cytomegalovirus (CMV) reactivation and disease is still a frequent complication after allogeneic stem cell transplantation (allo SCT). It is well accepted that T-cell immunity is mandatory to control CMV infection and disease and much effort has been put into the development of cell-based monitoring assays. Nevertheless, no reliable marker for protective immunity has been established to date. Most studies use one CMV model antigen (pp65) to compare the frequencies of cytokine producers (mainly IFNg) or multimer-specific T-cells. Methods: In total, we recruited 16 patients after allo SCT, (7 high risk, 9 standard risk pts.). We used 8-colour flow cytometry to detect degranulation (mobilized CD107a/b), intracellular IFNg, TNFa, IL-2 production and CD28-expression in peptide pool stimulated pp65 and IE-1 specific CD8 T-cells. Results were compared to 7 healthy CMV exposed donors. Results: Degranulation identifies the highest percentage of CMV-specific T-cells in allo-transplanted patients (pp65: 0,94% degranulation and 0,31% IFNg; IE-1: 1,44% degranulation and 0,87% IFNg, mean frequency). These T-cells are relatively cytokine deficient compared to those in healthy donors (cytokine-production/degranulation ratio: SCT=0,42, healthy=0,72 for pp65, p=0,048; SCT=0,61, healthy= 1,00 for IE-1, p=0,133, U-test). The cytokine expression pattern differs between antigens used for stimulation, for example more IL-2-producers could be detected in the pp65 specific compartment (12,5% for pp65 and 4,5% for IE-1 of all activated CD8 T-cells, p=0,015). Conclusion: This study demonstrates that degranulation is the most prominent marker of CMV-specific T-cells (pp65 and IE-1) in allo SCT patients. Looking at IFN-g producers only may underestimate the frequencies of CMV specific T-cells in this setting. Furthermore, these subsets have a divergent functionality in transplant recipients compared to healthy individuals. Our data challenge the concept of enumerating CMV specific T-cells to estimate immunity. We rather propose measuring functional differences in the T-cell response may help to identify patients with a high risk of CMV reactivation. A careful dissection of these differences is a prerequisite for the development of monitoring tools and adoptive T-cell transfer.

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