Reconstitution of the T Cell Repertoire Following Treatment with Alemtuzumab in Patients with B-Cell Chronic Lymphocytic Leukemia (B-CLL).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2986-2986
Author(s):  
Mohammad R. Rezvany ◽  
Mahmood J. Tehrani ◽  
Claes Karlsson ◽  
Jeanette Lundin ◽  
Hodjattallah Rabbani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Long Term Follow Up

Abstract Background and Methods: B-cell chronic lymphocytic leukemia (B-CLL) occurs as a result of clonal accumulation of functionally abnormal B cells. Alemtuzumab is a humanized monoclonal antibody specific for the CD52 antigen, which is highly expressed on both B-CLL cells and normal lymphocytes, but not on hematopoietic (CD34) stem cells. Alemtuzumab has been shown to effectively deplete the blood and bone marrow of lymphocytes, including CD4 and CD8 T cells, which may lead to profound immunosuppression and make patients more susceptible to infections. We and others have previously shown that the CD4 T cells in B-CLL patients may be clonally distinct from the normal population in that they present a more clonal pattern of the T-cell receptor (TCR) repertoire (Rezvany et al, Blood2003;101:1063–1070). It is therefore of interest to study the T cell repertoire following alemtuzumab administration as well as factors affecting T cell reconstitution following CD52 targeted therapy. In this study, we evaluated in depth the T-cell receptor-beta-variable sequence (TCR BV) in CD4 and CD8 T cells by real-time PCR, before and repeatedly after/during long term follow-up, in 5 B-CLL patients who had received alemtuzumab as first-line therapy (Lundin et al, Blood2002;100:768–773). Also, an analysis was conducted of CDR3 length polymorphism to describe changes in the clonality pattern. Results: A decline in most of BV genes either in CD4 or CD8 T cells was observed shortly after alemtuzumab treatment, which was followed by a gradual increase in most of the BV genes during long-term follow up. CDR3 length polymorphism analysis shortly after treatment revealed an even more highly restricted pattern in CD4 T cells compared to baseline with a shift towards a monoclonal/oligoclonal pattern regardless of increased or decreased BV usage. Furthermore, in the analysis of the clonal spectrum that was expressed shortly after alemtuzumab therapy, the number of peaks was significantly reduced in CD4 (P <0.01) but not in CD8 T cells, which was followed by a gradual increase in diversity towards a polyclonal repertoire during long-term follow up. Conclusions: These results indicate that perturbations in the T cell repertoire following alemtuzumab are complex, and are not reflected by changes in CD4/CD8 T cell numbers only. The restricted CDR3 pattern present prior to therapy became even more restricted after end of treatment, followed by a normalization of CDR3 patterns in CD4 T-cells during long-term follow-up. These results further suggest a regulatory role for T cells in relation to the malignant B cell clone in patients with B-CLL.

scholarly journals Characterization of human cytomegalovirus peptide–specific CD8+ T-cell repertoire diversity following in vitro restimulation by antigen-pulsed dendritic cells

Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 213-223 ◽  
Author(s):  
Karl Peggs ◽  
Stephanie Verfuerth ◽  
Arnold Pizzey ◽  
Jenni Ainsworth ◽  
Paul Moss ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Viral Replication ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Repertoire Diversity ◽  
Clonal Composition

Under conditions of impaired T-cell immunity, human cytomegalovirus (HCMV) can reactivate from lifelong latency, resulting in potentially fatal disease. A crucial role for CD8+ T cells has been demonstrated in control of viral replication, and high levels of HCMV-specific cytotoxic T-lymphocytes are seen in immunocompetent HCMV-seropositive individuals despite very low viral loads. Elucidation of the minimum portion of the anti-HCMV T-cell repertoire that is required to suppress viral replication requires further study of clonal composition. The ability of dendritic cells to take up and process exogenous viral antigen by constitutive macropinocytosis was used to study HCMV-specific T-cell memory in the absence of viral replication. The specificity and clonal composition of the CD8+ T-cell responses were evaluated using HLA tetrameric complexes and T-cell receptor β chain (TCRBV) spectratypic analyses. There was a skewed reactivity toward the matrix protein pp65, with up to 40-fold expansion of CD8+ T cells directed toward a single peptide-MHC combination. Individual expansions detected on TCRBV spectratype analysis were HCMV-specific and composed of single or highly restricted numbers of clones. There was preferential TCRBV gene usage (BV6.1/6.2, BV8, and BV13 in HLA-A*0201+ individuals) but lack of conservation of CDR3 length and junctional motifs between donors. While there was a spectrum of TCR repertoire diversity directed toward individual MHC-peptide combinations between donors, a relatively small number of clones appeared to predominate the response in each case. These data provide further insight into the range of anti-HCMV responses and will aid the design and monitoring of adoptive immunotherapy protocols.

Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells

10.1038/nm880 ◽  
2003 ◽  
Vol 9 (6) ◽  
pp. 727-728 ◽  
Author(s):  
Janet D Siliciano ◽  
Joleen Kajdas ◽  
Diana Finzi ◽  
Thomas C Quinn ◽  
Karen Chadwick ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Latent Reservoir ◽  
Follow Up Studies ◽  
Long Term Follow Up ◽  
The Stability ◽  
Hiv 1

T Cell Receptor Repertoire of Patients with Chronic Graft-Versus Host Disease.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5174-5174
Author(s):  
Olga Y. Azhipa ◽  
Scott D. Rowley ◽  
Michele L. Donato ◽  
Robert Korngold ◽  
Thea M. Friedman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Flow Cytometric Analysis ◽  
T Cell Responses ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Cell Responses ◽  
Flow Cytometric ◽  
Complexity Index

Abstract Chronic GVHD (cGVHD) is a major risk factor in patients receiving allogeneic hematopoietic cell transplantation (HCT), and is a complicated syndrome with a combination of autoimmune-like features and a range of multiorgan manifestations. Currently, efforts are being made to standardize the criteria for diagnosis and staging of cGVHD, but there is little understanding of the pathogenesis of the disease, associated biomarkers, and the immune perturbations that may result. Reconstitution of the T cell repertoire after allo-HCT often takes several months to a year, and may be significantly impaired or skewed in patients who develop cGVHD. We thus sought to assess the immune T cell status of cGVHD patients by TCR Vβ CDR3-size spectratype analysis. A cohort of 9 patients who underwent allo-HCT (PBMC n=7; BM n=2) were enrolled in the study. The underlying diseases in these patients were CML (n=1), AML (n=4), ALL (n=1), CLL (n=1), and MM (n=2). Patients received either reduced intensity or myeloablative conditioning before transplantation, and 8 of the 9 had a previous history of acute GVHD. Furthermore, the patients did not have evidence of infectious disease. PBMC was collected from each patient at one time point ranging from 2 wk to 3 yr from the time they were diagnosed with cGVHD. The onset of cGVHD ranged from 100 d to 3 yr post-HCT (median of 5 mo). Flow cytometric analysis was performed on peripheral blood lymphocytes from 7 of the 9 patients to analyze recovery of different subpopulations. PCR amplification of the CDR3 region of 21 TCR Vβ genes was used to analyze the diversity of the T cell repertoire. The PCR products were run on a sizing gel to separate the CDR3-lengths, and further analyzed by ABI GeneMapper software. Flow cytometric analysis revealed diverse percentages of CD4+ and CD8+ T cells among the 7 patients tested, which were correlated with the post-HCT period. Two patients who received HCT, 4 and 9 months before blood sampling, had only 3% and 4% CD4+ and 3% and 9% CD8+ T cells in their PBMC sample, respectively. On the other hand, the remaining 5 patients, who were all at later time points post-HCT, had CD4+ and CD8+ T cell percentages within normal range. One patient had a ratio close to the normal 2:1 CD4/CD8 ratio, two patients had a 1:1 ratio, and four had inverse CD4/CD8 ratios. Based on CDR3-size spectratype analysis, we determined the recipient TCR-Vβ complexity index within each resoluble family, which represented the percentage of the number of peaks found for each Vβ relative to that found in the average corresponding Vβ family of 10 healthy donors. We considered Vβ to be fully complex if the complexity index exceeded 85%. The results indicated that 41 to 88% of resolved Vβ in all 9 patients were fully complex, with the lower range corresponding to those patients sampled early post-HCT. Vβ 1, 2, 4, 6, 8, 12, and 13 families revealed the best recovery in all patients, even in patients after 4-mo post-HCT. Importantly, extensive skewing of the repertoire within most of the TCR Vβ families were found in all 9 recipients, suggesting that there were active heterogenous T cell responses in those patients with cGVHD. As to what these T cell responses were directed to remains to be seen, and could theoretically involve autoantigens, alloantigens, tumor antigens, or sub-detectable infectious agents. In any case, the presence of a wide-ranging T cell response in these patients may serve as an important new diagnostic indicator for cGVHD.

Evolution of the Donor T Cell Repertoire In Allogeneic Stem Cell Transplant Recipients In the Second Decade After Transplantation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 831-831
Author(s):  
Robert Q. Le ◽  
J. Joseph Melenhorst ◽  
Brenna Hill ◽  
Sarfraz Memon ◽  
Minoo Battiwalla ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
T Cell Receptor ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Cell Receptor ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Significant Difference

Abstract Abstract 831 After allogeneic stem cell transplantation (SCT), donor T lymphocyte immune function is slowly re-established in the recipient through reconstruction of the donor's post-thymic T cell repertoire and from T cell neogenesis in the thymus. Although long-term survivors from SCT appear healthy, their immune repertoire and differences from that of their donors have not been characterized. We studied 38 healthy patients surviving more than 10 years from a myeloablative SCT for hematological malignancy (median follow-up 12 years, range 10–16 years). T cell and natural killer (NK) cell repertoires in these patients were compared with cells from their stem cell donors cryopreserved at time of transplant and from the same donors at 10 year after SCT. The median age of both recipients and their sibling donors at time of transplant was identical (36 years). Patients received cyclosporine GVHD prophylaxis and delayed add-back of donor lymphocytes 30–90 days post transplant. Only one patient was on continued immunosuppressive treatment at the time of study. Compared with the donor pre-transplant counts there was no significant difference in the absolute lymphocyte, neutrophil, monocyte, CD4+ and CD8+ T cell, NK cell, and B cell subset counts. However, compared to their donors, recipients had a) significantly fewer naïve CD4+ and CD8+ T cells; b) lower T cell receptor excision circles levels; c) fewer CD4+ central memory T cells; d) more effector CD8+ T cells; e) and more FOXP3+ regulatory T cells. These data suggest that the patient had a persistent deficiency on T cell neogenesis. Molecular examination of the T cell receptor Vbeta (TCRBV) repertoire by spectratype analysis showed that there was no significant difference in total complexity score, defined as the sum of the number of discrete peaks for each Vbeta subfamily, between the patients and their donors. TCRBV subfamily spectratyping profiles of patients and donors, however, had diverged, with both gains and losses of peaks identifiable in both patient and donor. In conclusion, patients surviving 10 or more years after allogeneic SCT still show a T cell repertoire that reflects expansion of the donor-derived post thymic T cell compartment, with a limited contribution by new T cell generation and persistently increased Tregs. It therefore appears that a diverse TCRBV repertoire predominantly derived from the memory T cell pool is compatible with good health. Disclosures: No relevant conflicts of interest to declare.

T Cells Engineered with a Chimeric Antigen Receptor (CAR) Targeting CD19 (CTL019) Have Long Term Persistence and Induce Durable Remissions in Children with Relapsed, Refractory ALL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 380-380 ◽  
Author(s):  
Stephan A. Grupp ◽  
Shannon L Maude ◽  
Pamela Shaw ◽  
Richard Aplenc ◽  
David M. Barrett ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Research Funding ◽  
Dose Range ◽  
Single Chain

Abstract BACKGROUND CARs combine a single chain variable fragment (scFv) of an antibody with intracellular signaling domains. We have previously reported on CTL019 cells expressing an anti-CD19 CAR. Infusion of these cells results in 100 to 100,000x in vivo proliferation, durable anti-tumor activity, and prolonged persistence in pts with B cell tumors, including sustained CRs in adults and children with ALL (Grupp et al., NEJM 2013, Maude et al., NEJM 2014). We now report on outcomes and longer follow up of the first 30 pts with relapsed, refractory ALL treated on our pilot trial in pediatric ALL. METHODS T cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3ζ, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into children with relapsed or refractory CD19+ ALL. 26/30 pts received lymphodepleting chemotherapy the week prior to CTL019 infusion. The targeted T cell dose range was 107 to 108 cells/kg with a transduction efficiency of 11-45%. T cells for manufacturing were collected from the pt regardless of prior SCT status, not allo donors. RESULTS 30 children median age 10y (5-22y) with CD19+ ALL were treated. 25/30 pts had detectable disease on the day before CTL019 cell infusion, while 5 were MRD(-). A median of 3.6x106 CTL019 cells/kg (1.1-18x106/kg) were infused over 1-3 days. There were no infusional toxicities >grade 2, although 9 pts developed fevers within 24 hrs of infusion and did not receive a planned 2nd infusion of CTL019 cells. 27 pts (90%) achieved a CR, including a patient with T cell ALL aberrantly expressing CD19+. 3 did not respond. MRD measured by clinical flow cytometry was negative in 23 responding pts and positive at 0.1% (negative at 3 mo), 0.09%, 0.22%, and 1.1% in 4 pts. With median follow up 8 mo (1-26 mo), 16 pts have ongoing CR, with only 3 patients in the cohort receiving subsequent treatment such as donor lymphocyte infusion or SCT, 6-month EFS measured from infusion is 63% (95% CI, 47-84%), and OS is 78% (95% CI, 63-95%). CTL019 cells were detected in the CSF of 17/19 pts and 2 pts with CNS2a disease experienced a CR in CSF. 10 pts with a CR at 1 mo have subsequently relapsed, half with CD19(-) blasts. 2/5 pts who relapsed with CD19(-) disease had previously been refractory to CD19-directed blinatumomab and subsequently went into CR with CTL019. Figure 1 Figure 1. All responding pts developed grade 1-4 cytokine release syndrome (CRS) at peak T cell expansion. Detailed cytokine analysis showed marked increases of IL6 and IFNγ (both up to 1000x), and IL2R. Treatment for CRS was required for hemodynamic or respiratory instability in 37% of patients and was rapidly reversed in all cases with the IL6-receptor antagonist tocilizumab, together with corticosteroids in 5 pts. Although T cells collected from the 21 pts who had relapsed after allo SCT were median 100% donor origin, no GVHD has been seen. Grade 4 CRS was strongly associated with high disease burden prior to infusion and with elevations in IL-6, ferritin (suggesting macrophage activation syndrome) and C reactive protein after infusion. Persistence of CTL019 cells detected by flow cytometry and/or QPCR, and accompanied by B cell aplasia, continued for 1-26 months after infusion in pts with ongoing responses. QPCR showed very high levels of CTL019 proliferation, with all patients achieving peak levels >5000 copies/ug gDNA and 26 patients with peak levels >15,000 copies/ug gDNA. B cell aplasia has been treated with IVIg without significant infectious complications. Probability of 6-mo CTL019 persistence by flow was68% (95% CI, 50-92%) andrelapse-free B cell aplasia was 73% (95% CI, 57-94%). CONCLUSIONS: CTL019 cells can undergo robust in-vivo expansion and can persist for 2 years or longer in pts with relapsed ALL, allowing for the possibility of long-term disease response without subsequent therapy such as SCT. This approach also has promise as a salvage therapy for patients who relapse after allo-SCT with a low risk of GVHD. CTL019 therapy is associated with a significant CRS that responds rapidly to IL-6-targeted anti-cytokine treatment. CTL019 cells can induce potent and durable responses for patients with relapsed/refractory ALL; however, recurrence with cells that have lost CD19 is an important mechanism of CLT019 resistance. CTL019 therapy has received Breakthrough Therapy designation from the FDA in both pediatric and adult ALL, and phase II multicenter trials have been initiated. Disclosures Grupp: Novartis: Consultancy, Research Funding. Barrett:Novartis: Research Funding. Chew:Novartis: Research Funding. Lacey:Novartis: Research Funding. Levine:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Research Funding. Rheingold:Novartis: Consultancy. Shen:Novartis: Employment. Wood:Novartis Pharma: Employment. Porter:Novartis: managed according to U Penn Policy Patents & Royalties, Research Funding. June:Novartis: Research Funding, Royalty income Patents & Royalties.

Cyclophosphamide Plus Allogeneic CD4+ T Cell Infusion Induces Anti-Lymphoma Immunity Despite Lack of Graft-Versus-Host Disease (GVHD) or Sustained Engraftment.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3063-3063
Author(s):  
Sanju Jalla ◽  
Jie Wang ◽  
Leo Luznik ◽  
Ephraim J. Fuchs
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Immunity ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cd4 T Cell ◽  
Cell Leukemia ◽  
B Cell Leukemia ◽  
T Cell Help

Abstract Recent evidence suggests that tumor-bearing animals contain CD8+ T cells that can respond productively to a tumor vaccine, but that these T cells do not respond because of insufficient help from tumor-specific CD4+ T cells, which have either been inactivated or turned into anti-tumor suppressor T cells. We therefore devised a strategy to augment anti-tumor immunity by administering cyclophosphamide (Cy), to eliminate suppressor CD4+ T cells, followed by combining autologous tumor cell vaccination and infusion of partially MHC-mismatched, or haploidentical, CD4+ T cells as a source of T cell help for endogenous CD8+ T cells. Interestingly, the combination of Cy followed by haploidentical T cell infusion, with or without vaccine, induced potent systemic anti-tumor immunity resulting in cure of 40-50% of BALB/c mice harboring the A20 B cell leukemia/lymphoma. Depletion of CD8+ T cells from the infusate abrogated GVHD but did not compromise anti-tumor immunity. Allogeneic donor spleen cells that contained CD8+ T cells engrafted durably and caused lethal GVHD. In contrast, the combination of Cy plus CD8+ T cell-depleted spleen cell infusion induced only transient engraftment, peaking on day 7 and declining to undetectable levels by day 14. In the absence of Cy conditioning, allogeneic donor spleen cell infusions did not induce detectable chimerism beyond day 3. In summary, Cy plus allogeneic CD4+ T cell infusion induces potent anti-tumor immunity in a mouse model of B cell leukemia/lymphoma. Potential mechanisms of the therapeutic effect include direct tumor cytotoxicity by CD4+ T cells or allogeneic CD4+ T cell help for endogenous, tumor-specific CD8+ T cells.

Prospective Molecular Identification of Alloreactive CTL Clones in Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4972-4972
Author(s):  
Christine L. O’Keefe ◽  
Ronald Sobecks ◽  
Alexander Rodriguez ◽  
Julie Curtis ◽  
Elizabeth Kuckowski ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Molecular Analysis ◽  
Cd8 T Cells ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Activation Markers ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Donor Cells

Abstract The process of immune recovery after allogeneic HSCT can be characterized by an often profound oligoclonality of the TCR spectrum which may reflect: 1) A decreased diversity within the T cell population or 2) Expansion of individual clones that may be caused by specific antigenic drive exerted by pathogens (e.g., CMV) or alloantigens during the process of GvHD. Novel technologies based on the molecular analysis of the TCR repertoire can be applied to study clonal responses, including multiplex amplification of rearranged TCR VB chains followed by sequencing and quantitation of their contribution to the entire T cell repertoire. We initially studied the T cell repertoire after allogeneic HSCT in sibling (N=20) and matched unrelated (N=9) transplants. VB spectratyping was performed on CD8+ T cells in 22 patients; of the expanded VB families tested, 61.2% (30 of 49) were mono- or oligoclonal by genotyping. The clonal size and structure was determined by sequencing. Immunodominant clones contributed up to 5.4% (avg. 1.4%; range 0.1–5.4%) of all CD8+ T cells, indicating that certain stimuli may drive expansion of immunodominant clones. We originally hypothesized that these expanded clones were allospecific and likely played a role in GvHD; however, we found no correlation between the presence of significant expansions and grade III/IV GvHD. Therefore, in order to identify alloreactive CTL clones and their clonotypic markers, an alternative approach was devised. The proposed technique utilizes an allostimulation step: recipient cells serve as targets to induce activation of allospecific donor cells. Donor alloreactive cells are identified by their expression of activation markers, such as CD25 or CD69. After sorting, allospecific T cells are used as a source of cDNA for identification and quantitation of allospecific clonotypes. In this fashion, we have analyzed patients undergoing allogeneic sibling and matched unrelated donor grafting (N=7). Prior to transplant, allostimulation was performed and alloreactive CD8-derived clonotypes were subjected to molecular analysis. VB families represented within alloresponsive CTL populations that were oligoclonal by genotyping were subcloned and a large number of CDR3 clones were sequenced to identify the immunodominant clonotypes. Sequences have been derived from activated CD8+ donor cells in 6 cases; an average of 4 (range 1–7) VB families per pair have been characterized.. Although the presence of multiple VB families with a diversified CDR3 spectrum suggests the polyclonal nature of alloresponsive clones, immunodominant clones were identified. A total of 13 immunodominant clonotypes have been identified in 5 patients. Five such clones were identified in one donor/recipient pair; in each pair at least one immunodominant clonotype was isolated. Up to 18 clones per VB family were sequenced, and the average expansion contributed 56% to the entire VB family (range 15–100%). Clonotype-specific primers have been designed from two expanded clones and used to detect the allospecific clones in post-transplant blood samples in one patient/donor pair. In sum, molecularly defined marker clonotypes indicative of alloresponsive CTLs in HSCT can be individually and prospectively isolated. Such clonotypes may find application in tissue and blood diagnosis of GvHD.

T Cell Repertoire after Alpha/Beta-T Cell Depleted Allogeneic Hematopoietic Stem Cell Transplantation in Pediatric Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4582-4582
Author(s):  
Ivan Zvyagin ◽  
Olga Tatarinova ◽  
Ilgar Mamedov ◽  
Ekaterina Komech ◽  
Alexey Maschan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
T Cell Clones ◽  
Cell Clones ◽  
Tcr Repertoire ◽  
Repertoire Sequencing ◽  
Alpha Beta

Abstract Allogeneic transplantation of hematopoietic cells (HSCT) is an established method to treat different hematologic malignancies and disorders of hematopoietic and lymphoid system. Graft-versus-host-disease is one of the main risk factor for success of the procedure. Simultaneous depletion of alpha-beta T-cells and CD19+ cells in graft is the promising way to reduce the risk. The approach was recently introduced in clinical practice and many aspects of immune system reconstitution are still unknown. We applied improved technology for T cell receptor (TCR) repertoire sequencing to study origin and dynamics of T cell clones during 1 year follow-up period after allogeneic TCRαβ/CD19-depleted HSCT in children. We performed TCR repertoire sequencing for peripheral blood samples of patients before HSCT, at 2, 6 and 12 months after HSCT (n=21, 21, 17, 16 respectively), and for respective donor blood apheresis samples before abT/CD19 depletion. Twelve of the patients were diagnosed with acute leukemia and the others with non-malignant inherited and acquired blood disorders. For each patient data on recipient's T cell chimerism and counts of CD3+, naïve CD3+, alpha-beta T-cells and recent thymic emigrants (RTE) have been collected during 1 year follow-up period. Barcoding of each original TCR mRNA molecule passed to massive parallel sequencing allowed us to: (1) reduce sample preparation biases and quantitatively reconstruct of TCR repertoires; (2) equalize repertoire data analysis depth which is absolutely necessary for correct comparison of samples; (3) prevent risk of cross-contamination between samples and increase confidence of T clone origin determination. Two months after TCRαβ/CD19-depleted HSCT T cell repertoire mostly consists of several hundreds highly abundant clones. For patients with low recipient T cell chimerism from 13 to 504 largest T-cell clones (median 255, IQR 219, n=9, T cell chimerism <=20%) represented 80% of all T cells in peripheral blood. For comparison in healthy age-matched donors we found from 32,000 to 47,000 largest T-cell clones in identical analysis (median 43191, IQR 6493, n=14, data from Britanova O.V. et.al., JI 2014). The overall diversity at d60 after HSCT was also much less compared with the healthy subjects. We also found that most expanded T cell clones at d60 do not represent just a replica of the most expanded clones in graft samples, originating from low-abundant graft T cell clones. The diversity of T repertoire early after HSCT positively correlated with recipient T cell chimerism (the diversity was higher for those patients with higher percentage of recipient's T cells). Also patients with low chimerism had higher number of T clones originating from the graft than from d0 pre-transplant recipient repertoire in contrast to the patients with high T cell chimerism who had inverse ratio (median number of patient's clonotypes shared between graft and d0 was 56 or 3 for patients with low or absent chimerism (IQR = 24 or 19.25, n = 5) and 21.5 or 321.5 for patients with T cell chimerism >2% (IQR = 46.5 or 724.75, n = 10)). In addition CD4+ RTE count was higher for patients with high T cell chimerism. This observation was additionally confirmed by analysis of flow cytometry data for the expanded cohort of 105 patients at d60 after αβT-cell depleted HSCT (Wilcoxon rank sum test p-value = 0.002). Our results demonstrate that early after αβT-cell depleted HSCT repertoire of T cells are extremely skewed and unlikely able protect recipient efficiently. Observed recovery of T cell count mostly results from expansion of a few clones that have to divide intensely for the whole 60 days period in order to achieve the observed counts. Early reconstitution of TCR diversity and RTE counts in patients with substantial recipient T cell chimerism is mostly explained by surviving recipient T cells and intrathymic T cell progenitors, respectively. This work was supported by the Russian Science Foundation project №14-35-00105. Zvyagin I. is supported by grant MK-4583.2015.4. Disclosures No relevant conflicts of interest to declare.

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