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.

scholarly journals Association of Donor T Cell Repertoire in Host Lymphoid and Target Organs and Gvhd Development

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4525-4525
Author(s):  
Yongxia Wu ◽  
Jianing Fu ◽  
Anusara Daenthanasanmak ◽  
Hung D Nguyen ◽  
Mohammed Hanief Sofi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Clone ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
T Cell Clones ◽  
Cell Clones ◽  
Target Organs ◽  
T Cell Clone ◽  
Donor T Cells

Abstract The diversity and composition of T cell receptor (TCR) repertoire, which is the result of V, D and J gene recombination in TCR gene locus, has been found to impact immune responses in autoimmune and infectious diseases. The correlation of T-cell repertoire with the pathogenesis and outcome of graft-versus-host disease (GVHD) remain undefined. Here, by utilizing high-throughput sequencing of the gene encoding the TCRβ-chain, we comprehensively analyzed the profile of T-cell repertoire in host lymphoid and GVHD target organs after bone marrow transplantation (BMT). To understand whether T-cell repertoire is affected by different strength of alloantigen stimulation, we transferred same donor T cells derived from C57BL/6 (B6) mice into irradiated BALB/c (MHC-fully mismatched), B6D2F1 (MHC-haploidentical), BALB.b (MHC-matched ) and B6 recipients (syngeneic). Fourteen days later, T cells were isolated from recipient peripheral blood, spleen, peripheral lymphoid nodes (pLN), mesenteric lymphoid nodes (mLN), liver, lung, gut and skin for TCR sequencing. Clonality of donor T cells, which is inversely associated with TCR diversity, was significantly increased in either syngeneic or allogeneic recipients when compared with naïve donor T-cells, consistent with the concept that TCR diversity is reduced after T-cell activation and expansion. Increased TCR clonality was observed in lymphoid organs of allogeneic compared with syngeneic recipients, confirming that donor T cells were further activated in allogeneic recipients. However, decreased TCR clonality was observed in GVHD target organs of allogeneic compared with syngeneic recipients, suggesting that only limited donor T-cell clones were able to migrate in target organs in syngeneic compared to allogeneic recipients. The frequency of top clones in total productive rearrangements was increased in GVHD target organs especially liver of allogenic than syngeneic receipts. Interestingly, the frequency of top clones was positively associated with MHC disparity between donor and host, ranging from low to high in syngeneic, MHC-matched, haploidentical, and fully-mismatched recipients, respectively. To understand the extent to which TCR rearrangement is shared among different organs after BMT, we analyzed the overlap of TCR clones across different organs in the same recipients. T-cell clones were highly overlapping across organs, especially among GVHD target organs, in the same recipients after allogeneic BMT, although much lower overlapping in recipients after syngeneic BMT. The results suggest that alloantigen stimulation selectively activate certain T-cell clones and enrich antigen specific clones. On the other hand, much fewer shared clones were found among different recipients within the same group, regardless of MHC-disparity between donor and host. These results suggest that specific T-cell clones activated and expanded by alloantigens stimulation were highly different in individual recipients even with the same MHC-disparity between donor and host. Interestingly, the levels of clone overlapping were different in distinct organs among individual recipients. The level of T-cell clone overlapping was found high in liver of individual recipients regardless of the strength of alloantigen stimulation. The level of T cell clone overlapping was relatively high in pLNs and skin of the recipients after haploidentical BMT; whereas the level of T cell clone overlapping was relatively high in mLNs and gut of the recipients after MHC-matched BMT. These results suggest that skin may be a dominant target in haploidentical BMT and gut as a dominant target in MHC-matched BMT; whereas liver is a common target organ regardless. In conclusion, the current study establishes the association between MHC disparity, T-cell activation, and GVHD development in the level of donor T-cell repertoire. While TCR repertoire of donor T cells in peripheral blood or lymph nodes likely is representative in any individual recipient/patient, it is nearly impossible to identify T-cell clones that are pathogenic and shared among groups of recipients/patients even with the same MHC-disparity between donor and host. Disclosures No relevant conflicts of interest to declare.

scholarly journals The T cell repertoire for recognition of a phylogenetically distant protein antigen. Peptide specificity and MHC restriction of staphylococcal nuclease-specific T cell clones.

1986 ◽  
Vol 164 (3) ◽  
pp. 897-910 ◽  
Author(s):  
A Finnegan ◽  
M A Smith ◽  
J A Smith ◽  
J Berzofsky ◽  
D H Sachs ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Staphylococcal Nuclease ◽  
Protein Antigen ◽  
Cell Recognition ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
T Cell Clones ◽  
T Cell Recognition ◽  
Cell Clones

Previous studies (1) have indicated that the repertoire of murine T cells specific for a potentially complex protein antigen is in fact specific for a limited number of antigenic epitopes on that antigen in association with a given Ia molecule. Since those studies generally analyzed responses to antigens that differ in only a few amino acids from homologous murine molecules, it was possible that tolerance to self proteins was responsible for the limited T cell repertoire seen in responses to closely related proteins. It was therefore of interest to determine whether T cell recognition of a structurally and phylogenetically more distant protein molecule would also show specificity for a limited number of immunodominant peptides on that molecule. A series of experiments was designed to study the antigen fine specificity and MHC restriction of T cell clones specific for the bacterially derived antigen staphylococcal nuclease (Nase). T cell clones generated in (H-2b X H-2a)F1 (B6AF1) T cells were shown to be specific for Nase and to be restricted by either Ab alpha Ab beta or Ek alpha Ek beta. The fine specificity of these clones was then analyzed using cyanogen bromide and tryptic fragments and a series of overlapping 20-amino-acid synthetic peptides corresponding to and spanning the entire sequence of the Nase molecule. Two Ab alpha Ab beta-restricted clones were highly responsive to peptide 91-110, and not to other synthetic Nase peptides. In contrast, seven Ek alpha Ek beta-restricted clones were consistently responsive to peptide 81-100 and not to 91-110 or to other Nase peptides. Certain of these Ek alpha Ek beta-restricted T cells expressed an interesting crossreactivity, in that they responded to peptide 51-70 as well as to 81-100, although the response to 51-70 was characterized by a markedly shifted dose-response curve, indicating a reduced efficiency of activation by this peptide. Analysis of the amino acid sequences of these regions indicates that this unexpected crossreaction may have a structural basis. A single Nase-specific T cell line generated from BALB/c T cells was, in contrast to any of the B6AF1 clones studied, responsive only to peptide 61-80 and not to other peptides, including 81-100 or 91-110. Collectively, these findings show that Nase-specific T cells are responsive to discrete Nase peptides. Moreover, the present findings suggest that in T cell recognition of a complex and highly foreign protein antigen, a limited number of peptide epitopes are preferentially recognized by T cells in association with a given Ia molecule.

scholarly journals Antigen-reactive T cell clones restricted by mixed isotype A beta d/E alpha d class II molecules.

1990 ◽  
Vol 171 (2) ◽  
pp. 577-582 ◽  
Author(s):  
M Matsunaga ◽  
K Seki ◽  
T Mineta ◽  
M Kimoto
Keyword(s):  
T Cells ◽  
T Cell ◽  
Keyhole Limpet Hemocyanin ◽  
Class Ii ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
T Cell Clones ◽  
Cell Clones ◽  
Limiting Dilution ◽  
Limiting Dilution Cloning

Mixed isotype A beta dE alpha d class II molecule-restricted antigen-reactive T cell clones were obtained from (BALB/c x B6E alpha d)F1 mice. These T cell clones responded to keyhole limpet hemocyanin in the presence of (BALB/c x B6E alpha d)F1 but not CBF1 APCs. Both anti-A beta d and anti-E alpha mAbs blocked the proliferative responses of these clones. The frequency of such mixed isotype A beta E alpha-restricted T cell clones in (BALB/c x B6E alpha d)F1 mice was estimated to be approximately 10% from our limiting dilution cloning. The existence of such mixed isotype class II molecule-restricted T cells would have important implications for the expansion of the T cell repertoire as well as the induction of autoimmunity.

scholarly journals A minority of T cells recognizing tumor-associated antigens presented in self-HLA can provoke antitumor reactivity

Blood ◽  
2020 ◽  
Vol 136 (4) ◽  
pp. 455-467 ◽  
Author(s):  
Marthe C. J. Roex ◽  
Lois Hageman ◽  
Sabrina A. J. Veld ◽  
Esther van Egmond ◽  
Conny Hoogstraten ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Clone ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
T Cell Clones ◽  
Tumor Associated Antigens ◽  
Cell Clones ◽  
T Cell Clone ◽  
Self Antigens

Abstract Tumor-associated antigens (TAAs) are monomorphic self-antigens that are proposed as targets for immunotherapeutic approaches to treat malignancies. We investigated whether T cells with sufficient avidity to recognize naturally overexpressed self-antigens in the context of self-HLA can be found in the T-cell repertoire of healthy donors. Minor histocompatibility antigen (MiHA)-specific T cells were used as a model, as the influence of thymic selection on the T-cell repertoire directed against MiHA can be studied in both self (MiHApos donors) and non-self (MiHAneg donors) backgrounds. T-cell clones directed against the HLA*02:01-restricted MiHA HA-1H were isolated from HA-1Hneg/HLA-A*02:01pos and HA-1Hpos/HLA-A*02:01pos donors. Of the 16 unique HA-1H–specific T-cell clones, five T-cell clones derived from HA-1Hneg/HLA-A*02:01pos donors and one T-cell clone derived from an HA-1Hpos/HLA-A*02:01pos donor showed reactivity against HA-1Hpos target cells. In addition, in total, 663 T-cell clones (containing at least 91 unique clones expressing different T-cell receptors) directed against HLA*02:01-restricted peptides of TAA WT1-RMF, RHAMM-ILS, proteinase-3-VLQ, PRAME-VLD, and NY-eso-1-SLL were isolated from HLA-A*02:01pos donors. Only 3 PRAME-VLD–specific and one NY-eso-1-SLL–specific T-cell clone provoked interferon-γ production and/or cytolysis upon stimulation with HLA-A*02:01pos malignant cell lines (but not primary malignant samples) naturally overexpressing the TAA. These results show that self-HLA–restricted T cells specific for self-antigens such as MiHA in MiHApos donors and TAAs are present in peripheral blood of healthy individuals. However, clinical efficacy would require highly effective in vivo priming by peptide vaccination in the presence of proper adjuvants or in vitro expansion of the low numbers of self-antigen–specific T cells of sufficient avidity to recognize endogenously processed antigen.

scholarly journals OP0027 AS-RELATED TCR BETA CLONOTYPES ARE PRESENT IN DIFFERENT INFLAMED TISSUES OF PATIENTS WITH SPONDYLOARTHROPATHIES

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 14.3-15
Author(s):  
E. Komech ◽  
A. Barinova ◽  
E. Shmidt ◽  
T. Korotaeva ◽  
A. Koltakova ◽  
...  
Keyword(s):  
T Cells ◽  
Psoriatic Arthritis ◽  
Ankylosing Spondylitis ◽  
T Cell ◽  
Synovial Fluid ◽  
Intestinal Inflammation ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
T Cell Clones ◽  
Cell Clones

Background:Recently a group of T-cell clones with characteristic T-cell receptor (TCR) motif was identified in peripheral blood and synovial fluid of HLA-B*27+ patients with ankylosing spondylitis (AS) [1-2] - a prototypic disease from a wider group of spondyloarthropathies (SpAs). Extraarticular manifestations of AS could involve skin, intestine or eye. Emerging data indicate linkage between intestinal and joint inflammation, including expression of gut-associated integrins on synovial T-cells [3-4]. However, clonal T-cell composition and presence of identical clones in different inflamed sites in SpAs remains poorly studied.Objectives:To investigate clonal T-cell repertoire and presence of AS-related TCR motif in different sites of inflammation of patients with SpA.Methods:Samples of synovial fluid (SF) were obtained from HLA-B*27+ and HLA-B*27- patients with ankylosing spondylitis (AS) and psoriatic arthritis (PsA), as well as gut biopsy samples from patients with AS and Crohn’s disease (AS/CD) or ulcerative colitis (AS/UC), and conjunctival swabs from patients with uveitis (Uv) and with or without articular manifestations (Table 1). Also SF and gut biopsy samples were obtained from HLA-B*27+ patients with juvenile idiopathic arthritis (JIA). For one patient PsA patient paired samples of SF and gut biopsy were obtained.Table 1.Detection of the AS-related motif TRBV9_CASS[V/A/L/P][G/A] [L/T/V][F/Y]STDTQYF_TRBJ2-3 in bTCR repertoires of samples from different inflamed sites of patients with SpATissueDiagnosisB27+B27-AS-related TCR motif+ among all samples from B27+ donorsSynovial fluidAS2012PsAJIAIntestinal biopsyAS/CD433 / 4AS/UCJLAConjunctival swabUv804 / 8SF and gut samples were processed to isolate mononuclear cells, while conjunctival swabs were directly lysed in the lysis buffer. CD3+ β7-intergin+ cells were isolated from SF by fluorescence-activated cell sorting. Deep TCR repertoire profiling was carried out using UMI-based cDNA library preparation technology [1].Results:Identical T-cell clonotypes were detected between paired SF and gut samples of the same patient with psoriatic arthritis and intestinal inflammation. The subpopulation of β7-intergin+ SF T-cells shared significantly more identical clonotypes with gut biopsy repertoire compared to the bulk SF T-cell repertoire.Clonotypes belonging to the AS-related TCR beta motif TRBV9_CASS[V/A/L/P][G/A][L/T/V][F/Y]STDTQYF_TRBJ2-3 were detected in all inflamed tissues tested: synovial fluid, intestinal biopsies and conjunctival swabs of SpA patients (Table 1). Importantly, we observed these clonotypes exclusively in samples from HLA-B*27+ donors (n=26), but not in HLA-B27- context (n=15) with comparable analysis depth, thus confirming strong HLA-B*27-restriction of the clonotypes. The AS-related clonotypes were detected in the subpopulation of β7-intergin+ SF T-cells from HLA-B*27+ patients with PsA.Conclusion:For the first time we directly report the T-cell clonal sharing between synovial fluid and inflamed gut tissue of SpA patients. In sum our data suggests involvement of identical T-cell clones in inflammation in different anatomical sites in SpA.References:[1]Komech et al. Rheumatology (Oxford). 2018;57(6):1097-1104.[2]Faham et al. Arthritis Rheumatol. 2016;11(10):300-308.[3]Guggino et al.Ann Rheum Dis. Published Online First: 18 October 2019.doi:10.1136/annrheumdis-2019-216456.[4]Qaiyum et al Ann Rheum Dis. 2019;78(11):1566-1575.Acknowledgements:We thanks all the patients and medical personnel involved in the studyDisclosure of Interests:None declared

T Cell Repertoire Diversity After Umbilical Cord Transplant Predicts Mortality From Infection

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4202-4202 ◽  
Author(s):  
Colleen Delaney ◽  
Ryan O Emerson ◽  
Filippo Milano ◽  
Anna Sherwood ◽  
Adrienne Papermaster ◽  
...  
Keyword(s):  
T Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
T Cell Clones ◽  
Cell Clones ◽  
Tcr Repertoire ◽  
Repertoire Diversity ◽  
Equity Ownership

Abstract Abstract 4202 Background In a transplant study run at Fred Hutchinson Cancer Research Center, 34 patients with high risk hematological malignancies underwent a myeloablative procedure and were subsequently transplanted with double umbilical cord blood units. Peripheral blood samples were collected from each patient before myeloablation, and at 28, 56, 100, 180, and 360 days post-transplant. Methods At each time point, we used the immunoSEQ platform to perform high-throughput sequencing of rearranged T Cell Receptor (TCR) loci. Using immunoSEQ data, we were able to track the presence and frequency of individual TCR clones in each patient across time-points, as well as measuring the diversity of the TCR repertoire as a whole. We correlated our measure of TCR repertoire diversity with clinical outcomes in this cohort. Results The study produced two primary results. First, using the ability to track clones, the reconstituting TCR repertoire is shown to oscillate wildly with nearly complete turnover of the T cell repertoire occurring at least monthly after CB transplant. The largest T cell clones present in each blood draw drop below detection within weeks, contrasting with control data in which the top clones in healthy patients are not only observed in multiple subsequent time-points, but remain at high frequency. The second result is a test of the hypothesis that diversity of the T cell repertoire is a measure of immunocompetence, as a clinical application of high-throughput sequencing. Of the 34 patients, six died between Day 100 and Day 360 of non-relapse causes. At both Day 56 and Day 100, the diversity of the T cell repertoires of those six patients were far lower than the T cell repertoire diversity values of the remaining patients (P-value = 0.015). Conclusions We have demonstrated that the reconstitution of clinical immunity in cord blood transplantation patients is characterized by a highly unstable T cell compartment with very rapid turnover of T cell clones. Despite the transience of individual T cell clones, however, by two months after transplant T cell repertoire diversity as measured by high-throughput TCR sequencing accurately predicts risk of non-relapse mortality. Disclosures: Emerson: Adaptive Biotechnologies: Employment, Equity Ownership. Sherwood:Adaptive Biotechnologies: Employment, Equity Ownership. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties.

The Allo-HLA-DP Restricted T Cell Repertoire Contains a Variety of Tissue-Restricted Specificities with Therapeutic Value

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3356-3356
Author(s):  
Aicha Laghmouchi ◽  
Conny Hoogstraten ◽  
Peter Van Balen ◽  
Rick van de Water ◽  
Marian van de Meent ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Hematopoietic Cells ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
T Cell Clones ◽  
Carcinoma Cell Lines ◽  
Cell Clones ◽  
Therapeutic Value

Abstract T cell-depleted allogeneic stem cell transplantation (alloSCT) is applied in patients with hematological malignancies to reduce the risk of graft versus host disease (GvHD), but the associated increased risk of infections and disease relapse makes scheduled donor-lymphocyte-infusion (DLI) necessary. Since HLA-DP is not taken into account in the matching procedure, stem cell grafts from unrelated donors are often only 10/10 matched, and mismatched for HLA-DP. Under non-inflammatory conditions, expression of HLA-DP is restricted to hematopoietic cells. Therefore, treatment with HLA-DP mismatched donor CD4 T cells can induce a specific graft versus leukemia (GvL) effect. However, in some cases, patients receiving HLA-DP mismatched CD4 T cells suffer from GvHD mediated by a profound allo-HLA-DP specific immune response. Adoptive transfer of in-vitro selected allo-HLA-DP restricted donor T cells directed against antigens specifically expressed on hematopoietic cells may be an elegant strategy to induce a specific GvL effect without coinciding GvHD. To investigate the feasibility of this approach, the allo-HLA-DP restricted T cell repertoire was dissected to unravel potential tissue specificities and to investigate the presence of hematopoiesis-specific CD4+ T cells with therapeutic value within this compartment. To induce allo-HLA-DP directed T cell responses, HLA-DP mismatched (10/10 matched) donor/patient pairs were selected. Donor PBMC were co-cultured with patient mature monocyte-derived dendritic cells (DC) for 14 days. The donor cells were first re-stimulated with autologous DC and depleted of activated auto-reactive CD137+ T cells using magnet cell separation (MACS). The negative fraction was then stimulated with the HLA-DP mismatched patient DC to induce activation of allo-reactive T cells. These allo-reactive CD137+ CD4+ T cells were clonally isolated using flowcytometric cell sorting, and expanded. Allo-HLA-DP restricted recognition of different hematopoietic and non-hematopoietic stimulator cells by the T cell clones was assessed using IFNγ and IL-4 ELISA. The T cells were tested against a large panel of hematopoietic cells (monocytes, DC, EBV-LCL and PHA blasts) of donor and patient origin, leukemic cell samples (AML) and non-hematopoietic cells (IFNγ pretreated, HLA-class-II expressing, skin-derived fibroblasts, and target-HLA-DPB1-transduced HELA, lung, kidney, and colon carcinoma cell lines). After re-stimulation with patient DC, flowcytometry showed frequencies of 0.5-10% of allo-DC activated (CD137+) CD4+ T cells. After cell sorting 1521 T cell clones from 4 different HLA-DP mismatched patient/donor pairs were tested against donor EBV-LCL, donor DC, patient EBV-LCL and patient DC as initial screening for allo-reactivity. 80% of the T cell clones showed allo-reactivity, as defined by recognition of patient, but not donor-derived EBV-LCL and/or DC. 14% of the tested clones showed no reactivity and 6% were auto-reactive. The HLA-DP restriction was analyzed of 400 selected allo-reactive T cell clones, using a panel consisting of donor, patient and 3rd party EBV-LCL or DC, 3rd party fibroblasts and target HLA-DPB1 transduced HELA cells. Of these 400 T cell clones, 65% were confirmed to be HLA-DP restricted. From these allo-HLA-DP restricted T cell clones 80% recognized both hematopoietic and non-hematopoietic cells expressing the target allo-HLA-DPB1. The other 20% of the allo-HLA-DP restricted T cell clones only produced cytokines when stimulated with hematopoietic cells (EBV-LCL and/or DC), and not when stimulated with non-hematopoietic cells (Fibroblasts, HELA, carcinoma cell lines). Moreover, 40% of these T cell clones showing hematopoiesis-restricted recognition only recognized DC, but not EBV-LCL expressing the target HLA-DPB1 allele. These clones also recognized primary AML blasts and proliferating CD34+ progenitor cells, illustrating a myeloid lineage restricted recognition pattern. These results illustrate that reactivity of allo-HLA-DP restricted T cells is not only determined by the expression of the target HLA-DPB1 allele, but is also dictated by cell lineage-specific gene expression causing differential peptide expression in HLA-DPB1. As a result, a significant portion of the allo-HLA-DP specific T cell repertoire harbors a specific GvL recognition pattern with high therapeutic value. Disclosures No relevant conflicts of interest to declare.

scholarly journals Diversification of the antigen-specific T cell receptor repertoire after varicella zoster vaccination

2016 ◽  
Vol 8 (332) ◽  
pp. 332ra46-332ra46 ◽  
Author(s):  
Qian Qi ◽  
Mary M. Cavanagh ◽  
Sabine Le Saux ◽  
Hong NamKoong ◽  
Chulwoo Kim ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Chronic Infection ◽  
Cell Receptor ◽  
Control Mechanisms ◽  
Cell Repertoire ◽  
T Cell Clones ◽  
Varicella Zoster ◽  
Cell Clones

Diversity and size of the antigen-specific T cell receptor (TCR) repertoire are two critical determinants for successful control of chronic infection. Varicella zoster virus (VZV) that establishes latency during childhood can escape control mechanisms, in particular with increasing age. We examined the TCR diversity of VZV-reactive CD4 T cells in individuals older than 50 years by studying three identical twin pairs and three unrelated individuals before and after vaccination with live attenuated VZV. Although all individuals had a small number of dominant T cell clones, the breadth of the VZV-specific repertoire differed markedly. A genetic influence was seen for the sharing of individual TCR sequences from antigen-reactive cells but not for repertoire richness or the selection of dominant clones. VZV vaccination favored the expansion of infrequent VZV antigen–reactive TCRs, including those from naïve T cells with lesser boosting of dominant T cell clones. Thus, vaccination does not reinforce the in vivo selection that occurred during chronic infection but leads to a diversification of the VZV-reactive T cell repertoire. However, a single-booster immunization seems insufficient to establish new clonal dominance. Our results suggest that repertoire analysis of antigen-specific TCRs can be an important readout to assess whether a vaccination was able to generate memory cells in clonal sizes that are necessary for immune protection.

scholarly journals Clonal analysis of functionally distinct human CD4+ T cell subsets.

1988 ◽  
Vol 168 (5) ◽  
pp. 1659-1673 ◽  
Author(s):  
F T Rotteveel ◽  
I Kokkelink ◽  
R A van Lier ◽  
B Kuenen ◽  
A Meager ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Cd4 T Cell ◽  
Tnf Alpha ◽  
Target Cells ◽  
B Cell Differentiation ◽  
T Cell Clones ◽  
Cell Clones ◽  
Alpha Beta

A large number of CD4+ T cell clones, obtained from peripheral blood T lymphocytes by direct limiting dilution, allowed us to address the question whether functional heterogeneity exists within the human CD4+ T cell subset. Cytotoxic capacity of cloned T cells was analyzed with the use of anti-CD3 antibodies and target cells bearing FcR for murine IgG. 6 of 12 CD4+ clones obtained were able to lyse Daudi or P815 cells in the presence of anti-CD3 antibodies. The remaining six CD4+ T cell clones tested did not display anti-CD3-mediated cytotoxic activity and did not acquire this cytotoxic capacity during a culture period of 20 wk. In the absence of anti-CD3 mAb, no lytic activity against Daudi, P815, and K562 target cells was observed under normal culture conditions. Phenotypic analysis of these two distinct types of CD4+ T cells did not reveal differences with regard to reactivity with CDw29 (4B4) and CD45R (2H4) mAbs that have been described to recognize antigens associated with helper suppressor/inducer (respectively) CD4+ cells. The CD4+ clones without anti-CD3-mediated cytotoxic activities (Th2) consistently showed a high expression level of CD28 antigens, whereas the cytotoxic clones (Th1) expressed low amounts of CD28. Th1 CD4+ clones did produce IL-2, IFN-gamma, and TNF-alpha/beta, whereas the Th2 T cell clones produced minimal amounts of IL-2 and only low levels of INF-gamma and TNF-alpha/beta in response to anti-CD3 mAbs and PMA. Although not all CD4+ clones did release IL-4, there was no correlation with cytotoxic activity. Moreover, as compared with the Th1 CD4+ clones, Th2 CD4+ T cell clones proliferated moderately in response to immobilized anti-CD3 mAbs. However, proliferation reached the level of the cytotoxic clones when anti-CD28 mABs were present during culture. Both CD4+ subsets provided help for B cell differentiation upon stimulation with anti-CD3 mAbs. Our data suggest that the human CD4+ subset, in analogy to the murine system, comprises two functionally distinct T cell subpopulations, both of which are able to exert helper activity for polyclonal B cell differentiation, but which differ in cytotoxic capacity, lymphokine production, and requirements for proliferation. A function for these two types of T cells in the immune response is discussed.

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 &lt;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.

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