scholarly journals Characterization of the T-cell Repertoire after Autologous HSCT in Patients with Ankylosing Spondylitis

Acta Naturae ◽  
2018 ◽  
Vol 10 (2) ◽  
pp. 48-57
Author(s):  
E. A. Komech ◽  
I. V. Zvyagin ◽  
M. V. Pogorelyy ◽  
I. Z. Mamedov ◽  
D. A. Fedorenko ◽  
...  
Keyword(s):  
Ankylosing Spondylitis ◽  
T Cell ◽  
Peripheral Blood ◽  
Structural Changes ◽  
Clonal Diversity ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Hematopoietic Stem ◽  
The Impact ◽  
Autologous Hsct

Autologous hematopoietic stem cell transplantation (HSCT), a safer type of HSCT than allogeneic HSCT, is a promising therapy for patients with severe autoimmune diseases (ADs). Despite the long history of medical practice, structural changes in the adaptive immune system as a result of autologous HSCT in patients with various types of ADs remain poorly understood. In this study, we used high-throughput sequencing to investigate the structural changes in the peripheral blood T-cell repertoire in adult patients with ankylosing spondylitis (AS) during two years after autologous HSCT. The implementation of unique molecular identifiers allowed us to substantially reduce the impact of the biases occurring during the preparation of libraries, to carry out a comparative analysis of the various properties of the T-cell repertoire between different time points, and to track the dynamics of both distinct T-cell clonotypes and T-cell subpopulations. In the first year of the reconstitution, clonal diversity of the T-cell repertoire remained lower than the initial one in both patients. During the second year after HSCT, clonal diversity continued to increase and reached a normal value in one of the patients. The increase in the diversity was associated with the emergence of a large number of low-frequency clonotypes, which were not identified before HSCT. Efficiency of clonotypes detection after HSCT was dependent on their abundance in the initial repertoire. Almost all of the 100 most abundant clonotypes observed before HSCT were detected 2 years after transplantation and remained highly abundant irrespective of their CD4+ or CD8+ phenotype. A total of up to 25% of peripheral blood T cells 2 years after HSCT were represented by clonotypes from the initial repertoire.

scholarly journals Significant mobilization of both conventional and regulatory T cells with AMD3100

Blood ◽  
2011 ◽  
Vol 118 (25) ◽  
pp. 6580-6590 ◽  
Author(s):  
Leslie S. Kean ◽  
Sharon Sen ◽  
Olusegun Onabajo ◽  
Karnail Singh ◽  
Jennifer Robertson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Effector Memory ◽  
Cell Populations ◽  
Cell Repertoire ◽  
Hematopoietic Stem ◽  
Macaque Model ◽  
The Impact

AbstractIn this study, we used the rhesus macaque model to determine the impact that AMD3100 has on lymphocyte mobilization, both alone and in combination with G-CSF. Our results indicate that, unlike G-CSF, AMD3100 substantially mobilizes both B and T lymphocytes into the peripheral blood. This led to significant increases in the peripheral blood content of both effector and regulatory T-cell populations, which translated into greater accumulation of these cells in the resulting leukapheresis products. Notably, CD4+/CD25high/CD127low/FoxP3+ Tregs were efficiently mobilized with AMD3100-containing regimens, with as much as a 4.0-fold enrichment in the leukapheresis product compared with G-CSF alone. CD8+ T cells were mobilized to a greater extent than CD4+ T cells, with accumulation of 3.7 ± 0.4-fold more total CD8+ T cells and 6.2 ± 0.4-fold more CD8+ effector memory T cells in the leukapheresis product compared with G-CSF alone. Given that effector memory T-cell subpopulations may mediate less GVHD compared with other effector T-cell populations and that Tregs are protective against GVHD, our results indicate that AMD3100 may mobilize a GVHD-protective T-cell repertoire, which would be of benefit in allogeneic hematopoietic stem cell transplantation.

scholarly journals Quantitative assessment of T cell repertoire recovery after hematopoietic stem cell transplantation

2013 ◽  
Vol 19 (3) ◽  
pp. 372-377 ◽  
Author(s):  
Jeroen W J van Heijst ◽  
Izaskun Ceberio ◽  
Lauren B Lipuma ◽  
Dane W Samilo ◽  
Gloria D Wasilewski ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Quantitative Assessment ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Hematopoietic Stem

High-Throughput Sequencing Of T Cell Receptors Detects Signatures Of T Cell Repertoire That Predict MRD Status In Patients With Mantle Cell Lymphoma Undergoing Immunotransplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4251-4251
Author(s):  
Malek Faham ◽  
Joshua Brody ◽  
Holbrook E Kohrt ◽  
Debra K Czerwinski ◽  
Ronald Levy
Keyword(s):  
T Cell ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Cell Vaccine ◽  
Cell Transplant ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Blood Samples ◽  
Post Transplant ◽  
Before And After

Background A clinical trial is ongoing at Stanford for MCL patients in first remission that interdigitates an autologous CpG-stimulated tumor cell vaccine with autologous peripheral blood stem cell transplant (PSCT) (NCT00490529). In this trial, blood samples collected before and after vaccination and serially post-transplant are assayed for minimal residual disease (MRD) and for T cell repertoire using the LymphoSIGHT™ sequencing method (Faham et al., Blood 2012). We identified a set of T cell clones that appear to be responding to the vaccine, and therefore we investigated whether the number of these clonotypes was correlated with MRD status. Methods Using universal primer sets, we amplified rearranged IgH variable (V), diversity, and joining (J) gene segments from genomic DNA. Amplified products were sequenced to obtain >1 million reads. The B cell tumor-specific sequence was identified for each patient based on its high frequency in the original tumor biopsy. The presence of the tumor cells was then monitored in serial blood samples with a sensitivity of 1 cell per million leukocytes. The same blood samples were used for amplification, sequencing and analysis of the entire TCRβ repertoire. To facilitate identification of tumor vaccine-induced TCRβ clonotypes, we sequenced the TCRβ repertoire immediately before and after the administration of both the priming vaccination and a booster vaccination. We developed a metric called the vaccine response score (V score). This metric is calculated for each clonotype and reflects the increase in frequency after the initial vaccination AND after the boost. The formula for calculating V score is: V = F1 x F2 x square root [1/ (|F1 – F2| + 1)], where F1 and F2 represent the fold-change of the priming and boost vaccinations, respectively. Clonotypes with a V score >10 were deemed to be vaccination-induced by virtue of these frequency changes. Results In a series of 12 vaccinated patients, the number of clonotypes with V score ≥ 10 ranged between 0 and 262, with a median of 57. We utilized an antigen-specific analysis to validate that clones with high V scores (≥ 10) were in fact tumor-specific. For this analysis, we incubated peripheral blood mononuclear cells (PBMCs) with the tumor and then sequenced the TCRβ repertoire from cells obtained after culture. Clones that were enriched after culture compared to pre-stimulation PBMCs were deemed to be antigen-specific. These clones that are antigen-specific are highly likely to have a high V score compared to a random frequency-matched set of clones (P two tailed = 1.8 x 10-10), providing further evidence that clones with a high V score are tumor-specific. We then analyzed the relationship between V score and clinical outcome. Patients could be stratified into two groups with “high” (> 25) or “low” (<25) numbers of vaccine-responsive clonotypes. Patients in the high V score group, who had larger numbers of putative tumor-specific T cells, were more likely to have sustained molecular remission during the first-year post-transplant compared with patients in the low V score group (P = 0.018) (Figure 1). Conclusions T cell repertoire analysis identified clonotypes responding to the vaccination, and the presence of these vaccine-specific clonotypes correlates with MRD positivity at the important landmark of one year post-PSCT. Further analysis of additional patients enrolled on the MCL trial is ongoing. This data underscores the prognostic relevance of the sequencing-based V score metric and provides a novel approach for assessment of cancer immunotherapy responses. Disclosures: Faham: Sequenta: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Resetting of the Mucosal T Cell Repertoire after Hematopoietic Stem Cell Transplantation in Refractory Crohn’s Disease

2017 ◽  
Vol 152 (5) ◽  
pp. S613-S614 ◽  
Author(s):  
Lionel Le Bourhis ◽  
Ana Maria Corraliza ◽  
Claire Auzolle ◽  
Elena Ricart ◽  
Christopher J. Hawkey ◽  
...  
Keyword(s):  
Stem Cell ◽  
Crohn’S Disease ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Hematopoietic Stem

scholarly journals DOP084 Peripheral T cell repertoire reconstitution in Crohn's disease patients undergoing autologous HSCT

2017 ◽  
Vol 11 (suppl_1) ◽  
pp. S76-S76 ◽  
Author(s):  
A.M. Corraliza ◽  
M.C. Masamunt ◽  
E. Ricart ◽  
A. Lόpez-García ◽  
L. Le Bourhis ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
T Cell ◽  
Crohn's Disease ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Autologous Hsct

Clonality of T cell repertoire in the tumor (TME) and peripheral blood of regionally advanced melanoma patients (pts) treated with neoadjuvant ipilimumab (ipi) and high dose interferon-α (HDI).

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 9585-9585
Author(s):  
Priyanka Vallabhaneni ◽  
Tala Achkar ◽  
Marissa Vignali ◽  
Sharon Benzeno ◽  
Julie Rytlewski ◽  
...  
Keyword(s):  
T Cell ◽  
Peripheral Blood ◽  
Pathologic Complete Response ◽  
Advanced Melanoma ◽  
Cell Fraction ◽  
High Dose ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Primary Tumors ◽  
Significant Difference

9585 Background: Pts with regionally advanced melanoma were treated with neoadjuvant ipi and HDI in a reported study ( Tarhini. J Clin Oncol suppl, 2016; abstr 9585). Pathologic complete response (pCR) was found in 32% of pts. Clonality of T cell repertoire was investigated in TME and peripheral blood mononuclear cells (PBMC). Methods: Pts were randomized to neoadjuvant ipi 3 or 10 mg/kg combined with HDI. Tumor biopsies were evaluable for testing at pretreatment (N = 20 pts) and definitive surgery (week 6-8; N = 25). When available, primary (N = 24) and relapse tumors (N = 6) were tested. PBMC: pretreatment (N = 29), 6 weeks (wk) (N = 24), then 3 (N = 23), 6 (N = 21), 12 (N = 14) months. T cell receptor beta chain (TCRB) repertoire was immunosequenced in PBMC and TME to determine repertoire clonality and T cell fraction in blood and TME (TIL; fraction of all nucleated cells identified as T cells). Results: PBMC T cell fraction when measured early on-treatment (6 wks) was significantly higher in pts who had pCR or microscopic residual disease vs. gross disease at the 6-8 wks surgery (p = 0.047). PBMC clonality was significantly lower at 12 wks (p = 0.025) for pts who continued to be relapse free (NED) long term vs. those who eventually relapsed. In TME, except for trends no significant difference in clonality was seen, but in pts with pCR TIL fraction was significantly higher when measured in primary tumors (p = 0.033). The number of tumor-associated clones that were expanded in blood post-treatment was strongly correlated with both TIL fraction (Rho 0.7299, p = 0.0003) and TIL clone diversity (Rho 0.882, p = 2.7-7). Conclusions: Higher T cell fraction and lower clonality in PBMC when measured early on-treatment, and higher TIL fraction in primary tumor constituted promising biomarkers of response. Pts with higher TIL fractions were more likely to have tumor-associated clones detectable in blood, suggesting these may be useful for tracking the immune response. These findings warrant validation in an independent cohort and exploration with other immunotherapeutics. Clinical trial information: NCT01608594.

Clonal expansion of tumor infiltrating lymphocytes (TILs) in the peripheral blood of metastatic melanoma patients is significantly associated with response to CTLA4 blockade-based immunotherapy.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 2541-2541
Author(s):  
Arjun Khunger ◽  
Julie Rytlewski ◽  
Erik C. Yusko ◽  
Ahmad A. Tarhini
Keyword(s):  
T Cell ◽  
Clinical Response ◽  
Clin Oncol ◽  
Peripheral Blood ◽  
Clonal Expansion ◽  
Post Treatment ◽  
Response To Therapy ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Morisita Index

2541 Background: Patients with metastatic melanoma were treated on a clinical trial with tremelimumab and High Dose Interferon-Alfa (HDI) (Tarhini. J Clin Oncol. 2012). We previously reported that patients who achieved disease control and clinical response had significantly greater T-cell clonality (p = 0.0008) and T-cell fraction (p = 0.044) respectively in their pretreatment tumor biopsy samples (Tarhini. J Clin Oncol. 2017). In this study, we further characterize T-cell repertoire clonality and clonal expansion in the peripheral blood at different time points to evaluate the association between repertoire features and clinical response. Methods: Patients received tremelimumab 15 mg/kg I.V. every 12 weeks and HDI was given concurrently. Responses were assessed by RECIST as complete (CR) or partial (PR), stable disease (SD) or progression (PD). Peripheral blood mononuclear cells (PBMCs) from treated patients (N = 33) were obtained at baseline, day 29, and day 85 (following tremelimumab-HDI treatment); tumor samples at baseline were also obtained (N = 18). The T-cell receptor beta chain (TCRB) repertoire of PBMCs and tumor samples was immunosequenced using the immunoSEQ assay (Adaptive Biotechnologies), and repertoire clonality was assessed at baseline, day 29, and day 85. Differential abundance analysis was used to detect and quantify peripheral clonal expansion pre- versus post-treatment and identify the subset of peripheral clones also detected in the tumor repertoire. The Morisita Index of repertoire similarity was also calculated to compare global repertoire changes between pre- and post-treatment PBMC samples. Results: T-cell repertoire turnover, as measured by the Morisita Index, showed a trend towards responders (CR/PR) having greater turnover (lower Morisita Index) post-treatment than non-responders (SD/PD). Similarly, the total number of clones expanding in the peripheral repertoire varied over time within an individual (p = 0.034) but was not significantly affected by response to therapy (p = 0.275) or by on-treatment time point (p = 0.768). When the analysis was restricted to peripherally expanded clones that were also found in the tumor repertoire, responders had significantly more TILs expanded in the periphery at day 29 than non-responders (p = 0.036). Conclusions: Our analysis of the peripheral T-cell repertoire following treatment showed that detection of TILs in early peripheral clonal expansion correlates with response to therapy.

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.

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