Direct visualization of cytomegalovirus-specific T-cell reconstitution after allogeneic stem cell transplantation

Blood ◽  
2001 ◽  
Vol 97 (5) ◽  
pp. 1232-1240 ◽  
Author(s):  
Kate Cwynarski ◽  
Jenni Ainsworth ◽  
Mark Cobbold ◽  
Simon Wagner ◽  
Prem Mahendra ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Critical Role ◽  
Cell Immunotherapy ◽  
Unrelated Donors ◽  
Cmv Disease ◽  
Cmv Reactivation

Cytomegalovirus (CMV) remains an important cause of morbidity and mortality after allogeneic stem cell transplantation (SCT), but cytotoxic T lymphocytes (CTL) may play a critical role in controlling CMV reactivation. Fluorescent HLA-peptide tetramers containing immunodominant peptides from CMV were used to prospectively monitor the recovery of CMV CTL in recipients of allogeneic transplants from siblings (n = 13) or unrelated donors (n = 11). In patients given allografts from a sibling when both the patient and donor were seropositive for CMV before SCT, recovery of CMV-specific CTL was rapid and reached up to 21% of all CD8+ T cells. Early reconstitution of CMV-specific immunity was not observed if either the donor or recipient was seronegative for CMV. In recipients of transplants from volunteer unrelated donors, recovery of CMV-specific CTL was delayed in comparison to that in recipients of transplants from siblings and no CTL were observed within the first 100 days after SCT. CTL numbers were increased after episodes of CMV reactivation but were suppressed by prednisolone therapy. Recovery of CMV-specific CTL to levels greater than 10 × 106/L was associated with protection from CMV disease. It was concluded that use of HLA-peptide tetramers to quantify CMV CTL is valuable for studying T-cell responses after allogeneic SCT. It should allow prediction of CMV reactivation in individual patients and assist in the development of adoptive T-cell immunotherapy.

scholarly journals Clonal Dynamics and Publicness of CMV-Specific TCR Repertoire after Allogeneic Stem Cell Transplantation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4551-4551
Author(s):  
Takashi Toya ◽  
Ayumi Taguchi ◽  
Kazutaka Kitaura ◽  
Yuki Otsuka ◽  
Ryosuke Konuma ◽  
...  
Keyword(s):  
Stem Cell ◽  
Next Generation Sequencing ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Peripheral Blood ◽  
Tcr Repertoire ◽  
Cmv Reactivation ◽  
Generation Sequencing

[Background] Cytomegalovirus (CMV) disease is a major complication after allogeneic stem cell transplantation (SCT). However, mechanisms of adoptive immunity against CMV are not fully elucidated. Recently, high-throughput next-generation sequencing (NGS) technology made it possible to shed light on the detailed and comprehensive landscape of T-cell receptor (TCR) repertoire. In this study, we analyzed TCR repertoire of CMV-specific cytotoxic T-cells (CMV-CTLs) in patients who suffered from CMV reactivation after SCT to clarify the diversity and dynamics of CMV-specific T-cell immunity. [Methods] We sequentially collected peripheral blood mononuclear cells from patients with HLA-A*24:02 who received SCT in our institution. Samples were collected weekly or every two weeks from their neutrophil engraftment until approximately 100 days after SCT. CMV reactivation was evaluated weekly with CMV antigenemia test. CD8 and CMV pp65 tetramer positive cells were sorted and unbiased next-generation sequencing-based analyses of TRBV/TRBJ gene segments were performed at the timing of CMV reactivation in 16 patients, and TRA gene segments were also analyzed in 10 patients. In addition, TCR beta repertoires after 2-4 weeks were analyzed in 12 patients. In the 12 patients, the dynamics of TCR repertoire diversity and proportional changes of each clone were assessed. We evaluated the diversity by Shannon-Weber index, and we defined TCR beta clonotypes found in two or more patients using the same TRBV/TRBJ gene segments and CDR3 amino acid sequences as public. This study was approved by the ethics committee of Tokyo Metropolitan Komagome Hospital. [Results] Among 16 patients, 11 received bone marrow, 3 received peripheral blood stem cells, and 2 received cord blood transplant. Underlying diseases were acute myeloid leukemia in 7, acute lymphoblastic leukemia in 7, and myelodysplastic syndromes in 2 patients. Median age at SCT was 50 years old (range: 20-71). Median duration from SCT to first CMV reactivation was 39 days (range: 16-55) and 7 patients (43.8%) were administered systemic corticosteroid at the time of reactivation (prednisolone 10-30mg/day). Median peripheral blood CMV-CTLs count at that time was 29.47/uL (range: 4.65-229.6). In most patients TCR beta repertoire of CMV-specific CTLs when CMV reactivated was highly skewed (median Shannon-Weber index was 1.44 [range: 0.542-3.164]). TCR alpha and beta were analyzed together in 10 patients and their diversity correlated well (p<0.001). Interestingly, 12 of 16 patients (75.0%) had at least one public TCR and, in all 12 patients with public TCR, the most frequent TCRs were public. Diversity of TCR repertoire was significantly lower in patients with public TCR compared with those in patients without (p=0.039). There was no obvious association between presence of public TCR and multiple events of CMV reactivation (p=0.57). When we evaluated clonal transition in 12 patients whose TCR beta repertoires were analyzed sequentially, the public clonotypes were continuously detected in 10 of 11 patients with public TCR and remained dominant in 9, while in one patient frequency of the most frequent public clone declined massively (from 50.25% among assigned reads to 6.63%). In addition, there were two patterns of subsequent clonal behavior in TCR repertoire of CMV-CTLs. In 10 patients, prevailing clones persisted and TCR repertoires of CMV-CTLs remained oligoclonal (Figure A). However, in other two patients, TCR repertoires of CMV-CTLs became more diverse (Shannon-Weber index at the time of reactivation and a few weeks after was 0.558/2.958 in one patient and 1.471/3.86 in another), major clones lost, and new private clones appeared afterwards (Figure B). Polyclonal pattern (TCR repertoire of CMV-CTLs was diverse at the time of reactivation or after a few weeks) was detected in 3 patients out of 16 patients, and the pattern was exclusively seen in patients who were administered corticosteroid when CMV reactivated (42.9% vs 0.0%, p=0.063). [Conclusion] TCR repertoire of CMV-CTLs at the time of CMV reactivation after SCT is highly oligoclonal and frequently shared among different patients, but can dynamically change in a short period in some patients. Functional analyses of the dominant TCRs to understand their reactivity against CMV epitope and elucidation of the clinical significance and developmental mechanisms of clone shift are strongly warranted. Figure Disclosures Kitaura: Repertoire Genesis Inc.: Employment. Suzuki:Repertoire Genesis Inc.: Equity Ownership.

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.

Functional Helper T Cell Response to CMV Predicts Probability of CMV Vireamia Following Allogeneic Stem Cell Transplantation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2224-2224
Author(s):  
Hubertus C. Buyck ◽  
Samantha J. Paston ◽  
Mark W. Lowdell ◽  
Stephen Mackinnon ◽  
Vincent C. Emery
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Cell Response ◽  
T Cell Response ◽  
Reduced Intensity Conditioning ◽  
Cmv Reactivation ◽  
Reduced Intensity

Abstract CMV reactivation after allogeneic stem cell transplantation remains problematic and is increasing in frequency with the use of reduced intensity conditioning regimens. A functional immune response is critical to control viral replication and hence disease. In this study, 19 allogeneic stem cell transplant recipients at risk for CMV reactivation were followed for a median of 185 days. Six patients received myeloablative conditioning and thirteen patients received reduced intensity conditioning therapy. Functional CMV specific immune responses were monitored by flow cytometry, measuring CD4 and CD8 specific intracellular interferon gamma production in response to stimulation with peptide pools of 15 mer overlapping peptides spanning the entire length of the immunodominant PP65 and IE1 proteins. In addition the CD4 response to whole CMV antigen, which is capable of presenting multiple viral antigens, was also determined. 53% of patients experienced a CMV reactivation with a median time to 1st reactivation of 53.5 days (range 20–134). In comparison with the CMV reactivation group, the mean day 50 CD4 whole CMV antigen response was approximately 2 logs lower in the reactivated group (0.065X106/L) than the non-reactivated group (3.620X106/L) suggesting that CMV specific CD4 function is an important predictor of patients at risk of CMV reactivation. The incidence of CMV reactivation in patients receiving Campath-1H was 71% (10 out of 14 patients) while none of the 5 patients in the non-Campath-1H group reactivated. The use of Campath-1H as part of the conditioning regimen was associated with a significantly lower day 50 CD4 whole CMV antigen response (0.06X106/L) vs (4.53X106/L) for the non-Campath-1H group, suggesting a mechanism for the observed increased frequency of CMV reactivation with the use of this agent. The relative contribution of the CD4 and CD8 responses to IE1 and PP65 has also been determined in this patient group. In summary, the absolute functional helper T cell response to CMV was predictive of viral reactivation following transplant.

Variable Number of Tandem Repeat (VNTR) Disparity between Donor and Recipient Has Potential To Predict Outcomes of HLA-Identical Allogeneic Stem Cell Transplantation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5202-5202
Author(s):  
Dong Hwan Kim ◽  
Hee Doo Jung ◽  
Nan Young Lee ◽  
Joon Ho Moon ◽  
Sang Kyun Sohn ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Tandem Repeats ◽  
Chronic Gvhd ◽  
Critical Role ◽  
Variable Number ◽  
Chromosome 1 ◽  
Unrelated Donors

Abstract Background: Detecting the variable number of tandem repeats (VNTRs) between the recipient and the donor has already been adopted to monitor the degree of chimerism after allogeneic stem cell transplantation (SCT). In allogeneic SCT, besides MHC-disparity, the disparity of various polymorphous proteins encoded by several genes may play a critical role in the pathogenesis of graft-versus-host disease (GVHD). However, the biologic effect of VNTR disparity has scarcely been studied. Materials & Methods: Eighty-four patients receiving an SCT from HLA-identical sibling (n=68) or unrelated donors (n=16) were analyzed. The enrolled diseases included AML 48, ALL 8, CML 15, NHL 10, and high-risk MDS 3. A PCR was performed to amplify 3 VNTR regions (D1S80, D1S111, and D17S5). The disparity was classified as fully matched, partially matched, or mismatched pairs. Results: A strong correlation was observed between the D1S80 disparity and the transplant outcomes in terms of OS (p=0.0179) and NRM (p=0.0305), yet not for the D1S111 or D17S5 disparity. The fully matched D1S80 pairs showed a better OS (72% vs 38%) and lower NRM (17% vs 50%) compared to the partially matched or mismatched pairs. In multivariate analyses, a fully matched D1S80 pair was found to be an independent favorable prognostic factor for OS (p=0.03) and NRM (p=0.05). In addition, D1S80 disparity was significantly associated with the occurrence of gut chronic GVHD (p=0.05). Conclusion: The present data suggest that disparities in D1S80 - located in chromosome 1 - seemed to be associated with an increased incidence of gut chronic GVHD and NRM. Figure. Overall survial (A) and non-relapse mortality (B) according to D1S80 VNTR disparity Figure. Overall survial (A) and non-relapse mortality (B) according to D1S80 VNTR disparity

scholarly journals T-cell receptor repertoire of cytomegalovirus-specific cytotoxic T-cells after allogeneic stem cell transplantation

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Takashi Toya ◽  
Ayumi Taguchi ◽  
Kazutaka Kitaura ◽  
Fumi Misumi ◽  
Yujiro Nakajima ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Cytotoxic T Cells ◽  
Major Complication ◽  
Cell Receptor ◽  
Receptor Repertoire ◽  
Cmv Reactivation

AbstractCytomegalovirus (CMV) infection is a major complication during allogeneic stem cell transplantation (allo-SCT). However, mechanisms of adaptive immunity that drive this remain unclear. To define early immunological responses to CMV after transplantation, we using next-generation sequencing to examine the repertoire of T-cell receptors in CD8+/CMV pp65 tetramer+ cells (CMV-CTLs) in peripheral blood samples obtained from 16 allo-SCT recipients with HLA-A*24:02 at the time of CMV reactivation. In most patients, TCR beta repertoire of CMV-CTLs was highly skewed (median Inverse Simpson’s index: 1.595) and, 15 of 16 patients shared at least one TCR-beta clonotype with ≥ 2 patients. The shared TCRs were dominant in 12 patients and, two clonotypes were shared by about half of the patients. Similarity analysis showed that CDR3 sequences of shared TCRs were more similar than unshared TCRs. TCR beta repertoires of CMV-CTLs in 12 patients were also analyzed after 2–4 weeks to characterize the short-term dynamics of TCR repertoires. In ten patients, we observed persistence of prevailing clones. In the other two patients, TCR repertoires became more diverse, major clones declined, and new private clones subsequently emerged. These results provided the substantive clue to understand the immunological behavior against CMV reactivation after allo-SCT.

Sign in / Sign up

Export Citation Format

Share Document