Analysis of the Recent Thymic Output Function of 23 TCR Vβ Subfamily Naïve T Cells in Patients with AML.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4482-4482
Author(s):  
Qingsong Yin ◽  
Yangqiu Li ◽  
Lijian Yang ◽  
Shaohuao Chen ◽  
Xueli Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Output Function ◽  
Recent Thymic Emigrants ◽  
Thymic Output ◽  
Normal Individuals ◽  
Tcr Repertoire ◽  
Cellular Immune

Abstract Regeneration of the T-cell population proceeds normally along two different pathways, in which the thymic-dependent pathway accounts for the more durable reconstitution of the T-cell compartment, and generates a more diverse TCR repertoire. Thus thymus function serves as a direct index to understand cellular immune function and potential of long-term TCR Vβ repertoire reconstitution. The complexity of TCR repertoire is generated in the thymus by regular recombination of series of gene fragments of TCR α or β chains. During these process, by-products of rearrangements are generated in the form of signal joint T-cell receptor excision DNA circles(sjTRECs), as sjTRECs are stable extrachromosomal annular DNA, are not replicated while cell dividing, and their existence suggests functional TCR generation. Thus thymic function can be evaluated by measuring sjTRECs in peripheral blood. At present, the total recent thymic output function is evaluated by quantitating δRec-ψJα sjTRECs, but it can not evaluate particular thymic emigrants of different Vα or Vβ subfamily naïve T cells. As TCR Vα and Vβ naïve T cells include many subfamilies which play different immune roles, and the complexity of TCR repertoire is an important factor for cellular immune reconstitution. Our previous studies had showed that the recent thymic output function in patients with AML was significant decrease by quantitative detection of δRec-ψJα sjTRECs. To further estimate the recent thymic emigrants of different TCR Vβ subfamily naïve T cells, this study was designed to detect the existence of 23 TCR Vβ subfamily sjTRECs in peripheral blood mononuclear cells (PBMCs) by using 23 Vβ subfamily special primers (including 2Dβ1 sense primers and 23 Vβ subfamily antisense primers). TCR 23 Vβ-Dβ 1 sjTRECs were separately amplified in genomic DNA from 5×104 and 1×104 PBMCs of samples (10 cases of normal individuals and 32 cases of the different FAB subtypes of AML patients) to estimate the frequencies of TCR 23 Vβ-Dβ1 sjTRECs by using semi-nest PCR. The results indicated that the frequencies of 23 Vβ-Dβ1 sjTRECs were different at the same cellular concentration, and the higher cellular concentration the higher frequency of Vβ subfamily sjTRECs. The number of detectable Vβ subfamily sjTRECs was (5.09±3.28) and (2.59±2.06) in 5×104 and 1×104 PBMCs from AML patients, as compared with (13.7±2.67) and (5.50±2.07) from normal individuals, and the differences were significant (both p=0.000). About 5/23(22%) of Vβ-Dβ1 sjTRECs were detected in 5×104 PBMCs from AML patients, and the frequencies of 13 Vβ subfamily sjTRECs (including Vβ1,Vββ,Vβ3,Vβ4,Vβ5, Vβ9,Vβ10,Vβ12,Vβ13,Vβ14,Vβ17,Vβ22,Vβ24-Dβ1 sjTRECs) were significantly lower than those from normal individuals, among which the lowest were Vβ10 and Vβ14-Dβ1 sjTRECs, the most frequency was Vβ21. But the difference was not significant within the different FAB subtypes of AML patients. It was negative correlation between age and the number of detectable Vβ subfamily sjTRECs in patients with AML, and patients who were < 30 years tended to be higher number of detectable Vβ subfamily sjTRECs than those ≥ 30 years, which became significant at 5×104 PBMCs level (r=−0.481, p=0.005). Taken together, the recent thymic emigrants of 23 Vβ subfamily naïve T cells were absent to a different extent or lower level among patients with AML. These results suggested that AML patients had severe cellular immunodeficiency and the capacity and potential of long-term TCR Vβ repertoire reconstitution were dramatically lowered.

Detection of 24 TCR Vβ-Dβ1 sjTRECs in T Cells from Cord Blood, Peripheral Blood of Normal Individuals and Patients with AML-M2.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4557-4557
Author(s):  
Yangqiu Li ◽  
Qingsong Yin ◽  
Shaohua Chen ◽  
Lijian Yang
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Output Function ◽  
Thymic Function ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Thymic Output ◽  
Normal Individuals

Abstract Thymic function is characterized its importance of thymus to T-cell diversity in the periphery of both children and adults during both health and disease. The generation of TCR diversity occurs in the thymus through recombination of gene segments encoding the variable parts of the TCRα and β chains. During these processes, by-products of the rearrangements are generated in the form of signal joint T-cell receptor excision circles (sjTRECs). As sjTRECs are stable extrachromosomal DNA fragments, are not replicated during mitosis and thus diluted with each round of cell division, and are therefore most frequent in naïve T cells that have recently left the thymus, their quantification is actually considered as a very valuable tool to estimate thymic function. Quantitative of δRec-ψJα sjTRECs can direct evaluate the recent thymic output function, but it is unable to analyze the particular thymic output function of different TCR Vβ subfamily naive T cells. The complexity of TCR Vβ repertoire is an important factor for immune reconstitution, it should be established the quantitative analysis of series TCR Vβ-Dβ sjTRECs to evaluate the levels of different Vβ subfamily naive T cells. In the present study, analysis of 24 TCR Vβ-Dβ sjTRECs was established by semi-nested PCR using 24 Vβ subfamily antisense primers and 2 Dβ1 sense primers. TCR Vβ-Dβ sjTRECs were amplified in genomic DNA from mononuclear cells of 10 cord blood samples, 10 cases of peripheral blood from normol individuals and 11 cases with AML-M2. Different amounts of DNA (corresponding to 2*105, 5*104, 1*104 and 1*103 cells respectively) from all samples were amplified to estimate the frequency of TCR Vβ-Dβ sjTRECs. The results showed that most Vβ subfamily sjTRECs could be detected in all samples from cord blood and peripheral blood at 2*105 or 5*104 cells level, some of Vβ subfamily sjTRECs could be detected in 1*103 cells level. Whereas the frequency of Vβ subfamily sjTRECs were lower in peripheral blood T cells from patients with AML-M2 than in normal individuals (p<0.05). Vβ19, Vβ23 and Vβ24 subfamily sjTRECs could not be detected in all samples at 5*104 cells level. The results indicated that Vβ subfamily naive T cells could be detected with different frequency in peripheral blood of normaol individuals as well as in some patients with AML-M2. Lower frequency of Vβ subfamily naive T cells was found in most AML-M2 patients.

Changes in Thymic Recent Output Function in Patients with B-Cell Lymphocytic Malignancy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4464-4464 ◽  
Author(s):  
Yangqiu Li ◽  
Lijian Yang ◽  
Shaohua Chen ◽  
Suxia Geng ◽  
Grzegorz Przybylski ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Peripheral Blood ◽  
Cell Receptor ◽  
Hematologic Malignancies ◽  
Mean Value ◽  
Output Function ◽  
Thymic Output ◽  
Normal Individuals

Abstract Since 1998 the use of signal joint T cell receptor excision circles (sjTRECs) to study changes in the frequency of recent thymic emigrants was reported, this study drew attention to the fact that the level of sjTRECs can be used to estimate the recent thymic output function. Therefore, sjTRECs was used as a new marker for analysis of thymic output function in different immunodeficiency diseases, immune reconstitution in patients after stem cell transplantation. Defects of cellular immunity, however, may also play a role in hematologic malignancies. Little is known about the feature of T-cell immune state in B-cell lymphocytic malignancy. In order to identify number of naïve T-cells in B-cell lymphocytic malignancy, sjTRECs-content was determined in the mononuclear cell fraction and the sjTRECs-number was related to the number of T-cells (according to the number of CD3-positive cells). Quantitative analysis of sjTRECs in DNA of peripheral blood T cells from 45 cases with B-cell lymphocytic malignancy (including 17 cases with ALL, 4 cases with CLL, 15 cases with B-NHL and 6 cases with MM) were preformed by real-time PCR and TaqMan analysis, and 14 normal individuals as controls. The results showed a dramatic reduction of sjTRECs values in patients. Some cases no sjTRECs copies could be detected in 40000 T cells. The mean value of sjTRECs was 0.39±0.75 copies/1000 PBMCs in ALL, 0.11±0.15 copies/1000 PBMCs in B-CLL, 0.67±1.39 copies/1000 PBMCs in B-NHL, 0.91±1.16 copies/1000 PBMCs in MM patients, as compared with 4.1±3.65 copies/1000 PBMCs in normal individuals, the sjTRECs level in all goups of B-cells lymphocytic malignancy were significant decrease (p=0.0001, p=0.048, p=0.002) except MM group (p=0.053). However, when comparison of the sjTRECs value in CD3+ compartments, it was not statistically different from the sjTRECs counts between patients with ALL (3.91±7.20 copies/1000 CD3+cells) and normal individuals (6.36±5.28 copies/1000 CD3+cells) (p=0.2139), suggesting that the reduction of sjTRECs level in patients wit ALL may due to lower numbers of T cells in peripheral blood. In conclusions, the results suggest that some thymic dysfunction in T cell generation was detected at least in most patient with B-cell lymphocytic malignancy. However, whether this is due to clonal expansion of T-cells to antigens, or reflects impairment of immune function associated to the malignancy, remains an open question. In a prospective study sjTRECs levels will be determined in different T- cell compartments including CD45RA+, CD4+ and CD8+ cells and so on.

The Significant Decrease of Recent Thymic Output Function in Patients with Benzene-Poisoned Aplastic Anemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1338-1338
Author(s):  
Yangqiu Li ◽  
Sufang Han ◽  
Lijian Yang ◽  
Shaohua Chen ◽  
Yubing Zhou ◽  
...  
Keyword(s):  
T Cell ◽  
Aplastic Anemia ◽  
Peripheral Blood ◽  
Specific Treatment ◽  
Quantitative Detection ◽  
Output Function ◽  
Time Points ◽  
Thymic Output ◽  
Cell Counts ◽  
Normal Individuals

Abstract It has been known for many years that benzene causes hematotoxicity, as well as the toxicity in lymphopoiesis, and it associated with aplastic anemia and leukemia. The aim of the present study was to investigate the level of T-cell receptor excision DNA circles (TRECs) within peripheral blood mononuclear cells (PBMCs) in patients with benzene-poisoned aplastic anemia (AA), thereby to evaluate the content of naive T cells and the recent thymic output function. Quantitative detection of TRECs in DNA of PBMCs from 16 normal individuals and 7 cases with benzene-poisoned AA was preformed by real-time PCR using TaqMan technique. The results showed that TRECs level was 6.69±4.79/1000 PBMCs in normal individuals, it was significant decrease in patients with benzene-poisoned AA (2.24±1.57/1000 PBMCs, p&lt;0.05). The TRECs levels in 2 cases with benzene-poisoned AA were followed up in different time points up to 55 weeks. The TRECs level was persistent decrease after diagnosis of benzene-poisoned and leaving the benzene exposure workplaces and undergoing clinic specific treatment, even if peripheral blood cell counts were became normal levels. The TRECs levels were 1.35±0.87/1000 PBMCs (at 5 time points) and 0.61±0.45/1000 PBMCs (at 4 time pionts) in two cases respectively. The results indicated that the recent thymic output function was remarkable decrease in patients with benzene-poisoned AA. It may obviously damage the T cell immune function in benzene poisoning.

Detection of sjTRECs within Peripheral Blood T Cells Evaluation of Recent Thymic Output Function in Myelodysplastic Syndrome.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4919-4919
Author(s):  
Xin Du ◽  
Yang-qiu Li ◽  
Jian-yu Weng ◽  
Ze-sheng Lu ◽  
Rong Guo ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Output Function ◽  
Peripheral Blood Mononuclear ◽  
Thymic Output ◽  
Normal Individuals ◽  
Blood Mononuclear Cells ◽  
Immunological Abnormalities

Abstract Objective The myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem cell disorders, while, immunological abnormalities are frequently observed in patients with MDS. Several reports revealed that about 10% of MDS patients have clinical autoimmune disorders like skin vasculitis, rheumatic disease, or autoimmune hemolytic anemia. Furthermore, serological immunological abnormalities like hyper- or hypogammaglobulinemia, positivities of antinuclear antibody, positivities of direct Coombs test, or inverted CD4/8 ratios were found in 18–65% of patients with MDS. Recently immunosuppressive therapies including prednisolone, antithymocyte globulin, and cyclosporin A (CsA) are used to treat cytopenia in some patients with MDS. But it isn’t very clear the immunosuppressive mechanism in MDS and the value of the treatment. To analyze the content of signal joint Tcell receptor excision DNA circles signal joint T cell receptor excision DNA circles(sjTRECs) within peripheral blood mononuclear cells (PBMCs), thereby to infer the level of naive T cells and the recent thymic output function in patients with myelodyspoastic syndrom. Methods Quantitative detection of sjTRECs in DNA of peripheral blood mononuclear cells from 13 normal individuals and 8 patienets were performed by real-time polymerase chain reaction (PCR) and TaqMan technique. Results The median value of sjTRECs copies P1 000 PBMCs was 4.37±3.64 in normal individuals whereas it was1.07 ±1.40 copies P1 000 PBMCs in myelodysplastic syndrom patients (P &lt;0. 05). Conclusions MDS Patients decrease in recent thymic output function.

scholarly journals A Recurrent T-Cell Receptor γ Rearrangement of the Vγ9-JγP Type Represents the Major Clone of γδ T-Cells in Normal Individuals and Is Highly Suppressed in Lymphoproliferative Disorders

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1119-1119 ◽  
Author(s):  
Philippe Szankasi ◽  
Jonathan Schumacher ◽  
Olga Efimova ◽  
Todd W. Kelley
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Receptor ◽  
Peripheral Blood ◽  
Γδ T Cells ◽  
Cell Receptor ◽  
Lymphoproliferative Disorders ◽  
Normal Individuals ◽  
Tcr Repertoire

Abstract INTRODUCTION γδ T-cells expressing T-cell receptor (TCR) type Vγ9Vδ2 are well-established mediators of anti-tumor immunity and have demonstrable HLA-independent cytotoxic activity against both B- and T-cell non-Hodgkin lymphoma cells. Initial clinical trials using ex vivo expanded, adoptively transferred γδ T-cells to enhance tumor immunity have shown some promise but overall results have been lackluster. This indicates that wholesale polyclonal expansion prior to transfer is not likely to be effective. Thus more refined approaches are necessary. This requires a better understanding of their TCR repertoire. We recently developed a next generation sequencing (NGS)-based method for evaluating the spectrum T-cell receptor gamma (TRG) gene rearrangements present in clinical samples. In an effort to better understand the TCR repertoire of γδ T-cells we used this strategy to evaluate a series of samples from normal individuals and from individuals with B and T-cell lymphoproliferative disorders (LPDs). METHODS DNA was isolated from samples from 11 normal individuals (all peripheral blood, PB), 11 patients with a T-cell LPD (6 PB, 3 bone marrow; BM, 2 FFPE tissues), and 5 patients with a B-cell LPD (Hairy cell leukemia; HCL, 4 PB, 1 BM). γδ T-cells were sorted from the PB of 4 of the healthy donors by FACS. TRG rearrangements were PCR amplified using consensus primers and NGS libraries were prepared and sequenced on the Ion Torrent PGM platform. The data was analyzed as follows. NGS typically yielded up to 400,000 sequencing reads which were grouped by identical V, J, and CDR3 sequences into unique rearrangements (typically 15-30,000). The prevalence of a particular TRG rearrangement (or CDR3 sequence) was determined by the number of individual NGS reads with this unique sequence per the entire data set (percent of total reads). All rearrangements were then ranked by their prevalence. RESULTS We sequenced the TRG repertoire of isolated γδ T-cells from the peripheral blood of normal individuals (n=4). We found that a recurrent Vγ9-JγP rearrangement with the CDR3 sequence CALWEVQELGKKIKVF was always (4 of 4 samples) the most prevalent rearrangement in normal γδ T-cells (3.2-11.7-fold more prevalent than the second most common rearrangement; representing 4-11.9% of total reads). Similarly, analysis of a larger set of unsorted normal peripheral blood samples demonstrated high prevalence of the same canonical CDR3 in 5 out of 7 samples, confirming that it is very common in most individuals relative to all TRG rearrangements (among the top 10 most prevalent CDR3s in 4 of 5 samples). We also sequenced the TRG repertoire in 11 samples from patients demonstrating evidence of involvement by a T-cell LPD. Unexpectedly, all Vγ9-JγP type rearrangements were strongly suppressed in these samples including the one with the canonical CDR3. The canonical rearrangement (present in 6 /11 cases), or the most abundant Vγ9-JγP rearrangement when the canonical rearrangement was absent (absent in 5/11 cases), represented on average 0.036 ±0.024 % of all NGS reads in the samples from patients with T-cell LPDs compared to 0.72 ±0.72 % of total NGS reads in the normal controls. This represents on average a 19.9 fold reduction in the T-cell LPD samples. The median rank by abundance of the top Vγ9-JγP rearrangement dropped from 9th (normals) to 291st (T-cell LPD cases) indicating that the suppression was not simply a consequence of the presence of an abundant malignant T-cell clone in the data. The overall distribution of TRG V-segment usage in the normal and neoplastic samples was comparable (Vγ9: 9.9 ±0.55 % and 7.2 ±2.79 %, respectively). In 2 samples from patients with HCL, Vγ9-JγP rearrangements were reduced to a similar extent to that seen in the T-cell LPD cases (9.5-fold reduced; average 0.06% of NGS reads; average rank order 451st). In the other 3 cases of HCL the findings were very similar to those seen in the normal samples. CONCLUSIONS We identified a recurrent Vγ9-JγP rearrangement by NGS representing the most abundant CDR3 in sorted γδ T-cells from normal individuals. This population, along with other clones with Vγ9 rearrangements, appeared specifically suppressed in all samples from patients with T-cell LPDs and in 2 or 5 samples from patients with B-cell LPDs (HCL), perhaps indicating a role in the disease process. Additional samples from a wider range of T- and B-cell LPDs are being analyzed. Disclosures No relevant conflicts of interest to declare.

scholarly journals A new method for the detection of Lesch-Nyhan heterozygotes by peripheral blood T cell culture using T cell growth factor

Blood ◽  
1984 ◽  
Vol 63 (4) ◽  
pp. 912-916 ◽  
Author(s):  
N Kamatani ◽  
H Yamanaka ◽  
K Nishioka ◽  
T Nakamura ◽  
K Nakano ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Growth Factor ◽  
Cell Growth ◽  
Peripheral Blood ◽  
Normal Individuals ◽  
T Cell Growth ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
T Lymphoblasts ◽  
T Cell Growth Factor

Abstract Thioguanine-resistant T lymphoblast populations were selectively amplified using T cell growth factor in the cultures of peripheral blood T cells from four Lesch-Nyhan heterozygotes. Although Lesch-Nyhan T lymphoblasts were all thioguanine-resistant, none of the cultures from 13 control subjects yielded the growth of such defective cell populations. These data provide direct evidence for the existence of a small percentage (5%–40%) of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient T cells in the heterozygotes, but not in normal individuals. Conversely, culture of the T lymphoblasts with azaserine plus hypoxanthine permitted the growth of the other part of the cell population that was enzyme positive. The low percentages of HGPRT-negative cells among T cells in heterozygotes suggest that the presence of this enzyme is beneficial for differentiation of lymphocytes of T cell linkage. Considering the ease and the reliability, culture of the peripheral T cells with thioguanine and T cell growth factor is very likely of practical use for detecting Lesch-Nyhan syndrome carriers among predisposed females.

Cellular Immune Responses To Vector In a Gene Therapy Trial For Hemophilia B Using An AAV8 Self-Complementary Factor IX Vector

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 717-717
Author(s):  
Etiena Basner-Tschakarjan ◽  
Federico Mingozzi ◽  
Yifeng Chen ◽  
Amit Nathwani ◽  
Edward Tuddenham ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Factor Ix ◽  
High Dose ◽  
Advisory Committees ◽  
Ifn Γ ◽  
Cellular Immune

Abstract In a clinical study of gene transfer for hemophilia B an adeno-associated virus vector serotype 8 (AAV8) expressing a self-complementary liver-specific expression cassette for the factor IX (FIX) transgene was administered intravenously in ten affected subjects. The results of the first part of the study have been published (NEJM 365:2357-65, 2011). In this abstract we present the immunomonitoring data, using Interferon-gamma (IFN-γ) ELISpot and polyfunctional T cell analysis of peripheral blood mononuclear cells (PBMCs) to monitor cellular immune responses to vector capsid and to Factor IX. We have previously shown that the cellular immune response was directed solely towards AAV capsid epitopes, not FIX, and that the response was dose-dependent. Out of six subjects infused in the high dose cohort (2x1012vg/kg), 4/6 manifested a minor rise in liver enzyme levels and detection of capsid-specific T cell reactivitiy in the ELISpot assay at ∼7-10 weeks post vector infusion. Maximum results on IFN- γ ELISpots ranged from 200-500 sfu/million cells. In two of these cases a modest decline in FIX level also occurred. Prompt initiation of prednisolone reversed these effects and rescued FIX levels. The remaining two subjects infused at the high dose, showed no rise in liver enzyme levels at any time point. However capsid reactive T cells were detectable in one subject as early as one to two weeks after vector infusion in peripheral blood by IFN-γ ELISpot assay, while no activation at all was detected in the other subject, possibly due to low cell recovery and viability of the cells. A similar immune response profile, with early detection of activated T cells but no rise in liver enzymes, was also observed in both subjects in the intermediate dose cohort in the first part of this study. Polyfunctional T cell analysis revealed concurrent Interleukin-2, Tumor necrosis factor-alpha and CD107a positivity in activated T cells at the peak of activation. Furthermore it showed that capsid-specific early T cell responses were detectable in the CD4+ T cell and later in the CD8+T cell compartment. Long-term immune monitoring of all subjects is ongoing. Importantly in one of the first two subjects treated at the high dose, capsid reactive T cells were detected by ELISpot 1.5 years after gene transfer; these cells were not detected in the other subject in whom long-term follow-up samples are available. Of note, capsid-reactive T cells were also seen at late time points (>1 year after infusion) in a middle dose subject and a low dose subject. Despite detectable T cell reactivity towards the AAV capsid in the peripheral blood FIX expression remained stable, suggesting that there is a short window of time during which transduced hepatocytes present a target for cytotoxic T cells, and that T cell positivity after this window is without any clinical consequences. In conclusion, for this scAAV8 vector there appears to be a critical threshold vector dose for a clinically detectable immune response, starting at 2x1012 vg/kg. The clinically detectable response occurred in four out of six subjects so far, and was manifest within a critical time interval of 7-10 weeks post infusion. The capsid-specific response was polyfunctional and detected in CD4+ and CD8+T cells in peripheral blood. It is important to note that not all subjects treated at the high dose developed an immune response. However, given the limited dataset, it is not yet possible to define predictive parameters, e.g. HLA type of a subject, for an immune response. Continued monitoring and future studies with more subjects will be necessary to confirm the presented findings, in particular time and rate of occurrence of a cellular response as well as successful treatment with a short course of Prednisolon. Disclosures: Tuddenham: Pfizer: Consultancy. Reiss:Hemophilia of Georgia: Honoraria. High:BristolMyersSquibb: Consultancy, membership on a Data Safety and Monitoring Board, membership on a Data Safety and Monitoring Board Other; Elsevier, Inc.: royalties from textbook, royalties from textbook Patents & Royalties; Genzyme, Inc.: Membership on an entity’s Board of Directors or advisory committees; Intrexon: Consultancy; Novo Nordisk: Consultancy, Member of a grant review committee, Member of a grant review committee Other; Shire : Consultancy; Benitec: Consultancy; bluebirdbio, Inc.: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; BioMarin: Consultancy; Alnylam Pharmaceuticals: Consultancy, Membership on an entity’s Board of Directors or advisory committees.

Distinct contributions of CD4+ and CD8+naive and memory T-cell subsets to overall T-cell–receptor repertoire complexity following transplantation of T-cell–depleted CD34-selected hematopoietic progenitor cells from unrelated donors

Blood ◽  
2002 ◽  
Vol 100 (5) ◽  
pp. 1915-1918 ◽  
Author(s):  
Matthias Eyrich ◽  
Tanja Croner ◽  
Christine Leiler ◽  
Peter Lang ◽  
Peter Bader ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Progenitor Cells ◽  
T Cell Receptor ◽  
Peripheral Blood ◽  
Cell Receptor ◽  
T Cell Subsets ◽  
Memory T Cell ◽  
Tcr Repertoire ◽  
Unrelated Donors

Normalization of restricted T-cell–receptor (TCR) repertoire is critical following T-cell–depleted (TCD) stem cell transplantation. We present a prospective study analyzing respective contributions of naive and memory T-cell subsets within the CD4+ and CD8+ compartments to the evolution of overall TCR-repertoire complexity following transplantation of CD34-selected peripheral blood progenitor cells from unrelated donors. During the first year after transplantation, sorted CD4/45RA, CD4/45R0, CD8/45RA, and CD8/45R0 subsets were analyzed at 3-month intervals for TCR-repertoire complexity by CDR3 size spectratyping. Skew in TCR-repertoire was observed only in early memory-type T cells. CD4+ and CD8+ subsets differed in clonal distribution of CDR3 sizes, with rapid Gaussian normalization of bands in CD4/45R0+ T cells. Naive T cells displayed normal repertoire complexity and contributed significantly to skew correction. Our data provide direct evidence for an important role of de novo maturation of naive T cells in normalization of an initially restricted TCR-repertoire following transplantation of CD34-selected, TCD-depleted peripheral blood progenitors from unrelated donors.

scholarly journals The T-Cell Receptor Repertoire Predicts Interim-PET in Patients with DLBCL Treated with R-CHOP: An Observational Study from a Prospective Clinical Trial

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 825-825
Author(s):  
Mohamed Shanavas ◽  
Mark Hertzberg ◽  
Rodney J Hicks ◽  
John F Seymour ◽  
Joshua W.D. Tobin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Peripheral Blood ◽  
T Cell Responses ◽  
Cell Receptor ◽  
Variable Degree ◽  
Cell Responses ◽  
Interim Pet ◽  
Tcr Repertoire

Abstract T-cell infiltration of the tumor microenvironment (TME) in DLBCL is a key determinant of response to chemo-immunotherapy (Keane, Lancet Haem 2015). We have previously shown that greater diversity of the T-cell receptor (TCR) repertoire within the TME is correlated with improved survival following R-CHOP in DLBCL (Keane, CCR 2017). There are limited data on the impact of the intratumoral TCR repertoire on interim-PET (iPET), the relationship between intratumoral and circulating TCRs, and on dynamic changes of the TCR during therapy. In this study, we interrogated the TCR repertoire in a subset of DLBCL patients treated on the prospective Australasian Leukaemia Lymphoma Group NHL21 study (Hertzberg, Haematologica 2017), in which all patients had 4x RCHOP prior to iPET risk stratification. The CDR3 region of TCRβ chain underwent high-throughput unbiased TCRβ sequencing (Adaptive Biotechnologies). Metrics included: productive templates (total functional T-cells), productive rearrangements (functional T-cells with distinct specificity), productive clonality (repertoire unevenness due to clonal expansions), and maximal frequency clones (% most dominant single clone). Matched intratumoral diagnostic samples, blood at pre-therapy and post-cycle 4 (at the time of iPET) were tested. 42 patients (enriched for iPET+ cases) had sufficient material for testing. Median age was 55 (range 22-69) years and 72% were males. IPI was low/intermediate/high in 13/63/25% respectively. Cell of origin (COO) by Lymph 2CX method (nanoString) was ABC in 30%, and GCB in 44%. 40% were iPET+. In tissue, there was a median of 4652 productive templates, translating into 2998 productive rearrangements identified. Notably, the clonal repertoire of intratumoral TCRs in iPET+ patients was larger than iPET-ve patients (productive clonality 8.1 vs 5.1 x10-2, p=0.04), whereas the numbers of functional T-cells did not vary between groups. Comparing the tumor with the blood samples showed a high, but variable, degree of overlap between peripheral blood and the TME - TCR repertoire. Median number of top 100 tumor tissue clones shared in peripheral blood was 53.5 (range, 1-97) in pre-therapy and 39.5 (range, 0-93) in post-therapy blood, indicating that the both the circulation and the tumor likely contribute to immune-surveillance. In pre-therapy blood, the median productive templates and productive rearrangements were 44,950 (range, 6,003-273,765) and 29,090 (range, 5,190-152,706), and the median clonality was 8.5 (1.46-45.3) x 10-2. There were no differences between iPET+ and iPET-ve patients in these parameters. However, there was a marked change in T-cell composition between time points. Interestingly, in iPET-ve patients clonality measures were increased, with productive clonality 9.4 vs 14.4 x10-2, p=0.03; and % maximum productive frequency 3.39 vs 5.89, p=0.04. These findings demonstrate that the intratumoral TCR repertoire, and sequential blood sampling provide important information on outcome in DLBCL treated with RCHOP. A highly clonal T-cell repertoire in the TME was associated with iPET positivity after 4 cycles of R-CHOP. In line with findings in solid cancers treated with checkpoint blockade, development of clonal responses in peripheral blood was associated with iPET negativity. These findings indicate that clones expanded during therapy may be important in tumor clearance but that highly clonal T-cell responses in the tumor at diagnosis may hinder expansion of other T-cell responses to neoantigens. The circulating TCR composition is representative of the TME. These findings will assist the rationale design and therapeutic monitoring of novel immuno-therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Peripheral T cell activation in long-term renal transplant patients: concordant upregulation of adhesion molecules and cytokine gene transcription.

1996 ◽  
Vol 7 (11) ◽  
pp. 2476-2482 ◽  
Author(s):  
F Kern ◽  
S Ode-Hakim ◽  
H Nugel ◽  
K Vogt ◽  
H D Volk ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Renal Transplant ◽  
Peripheral Blood ◽  
Cmv Infection ◽  
Cytotoxic Potential ◽  
Granzyme A ◽  
Transplant Patients ◽  
Renal Transplant Patients

In renal transplant, patients, the number of T cells expressing high levels of LFA-1 (LFA-1-bright) and of T cells expressing CD57 increases in response to viral infection, even if the latter is asymptomatic. Their role in long-term renal transplant patients with cytomegalovirus (CMV) antigenemia and concomitant transplant dysfunction was investigated. For this purpose, this study used triple-color flow cytometry, fluorescence-activated cell sorting of peripheral blood T cells (CD3+/LFA-1-dim or -bright and CD8+/CD57+ or CD57- subsets), and subsequent semiquantitative reverse transcription-polymerase chain reaction. Cytokine mRNA levels for interleukin (IL)-1 beta, IL-2, IL-4, IL-8, IL-10, tumor necrosis factor alpha, and interferon-gamma, as well as Granzyme A and IL-2R p55 and p75 transcripts were determined and compared in peripheral blood mononuclear cells and in separated T cell subsets. Although in patients with CMV infection and/or rejection, cytokine transcripts were readily detected and the levels in the CD3+/LFA-1-bright subsets were, by orders of magnitudes, higher than in the LFA-1-dim subset, hardly any cytokine message was found in patients without CMV infection or rejection episodes or in control subjects. The expression of Granzyme A, which is involved in cytotoxic T lymphocyte-mediated cytotoxicity, was not upregulated in LFA-1-bright T cells, which is in discordance with cytokine levels. Differences between CD57+ and CD57- T cells were limited to the IL-2R p55 mRNA, of which the former expressed significantly less than the latter. It is concluded that upon virus-induced activation of peripheral blood T cells, an effector type that is marked by high inflammatory but small cytotoxic potential is produced. The results of this study propose that these cells represent a correlate of persistent immune activation and are liable to produce graft dysfunction, although they are unable to clear the organism from virus infection because of their lack of cytotoxic potential.

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